Theranostic Bionanomaterials 0128153415, 9780128153413

Table of contents :
Cover
Theranostic Bionanomaterials
Copyright
List of Contributors
About the Editors
Part I: Fundamentals of Theranostic Bionanomaterials
1 Biodistribution, Excretion, and Toxicity of Inorganic Nanoparticles
Abbreviations
1.1 Introduction: Inorganic Nanoparticles and Their Interest in Medicine
1.2 Physicochemical Modifications of Inorganic Nanoparticles in Physiological Environments Determine Their Effects: Safety ...
1.2.1 Effects of the Agglomeration and Aggregation of Nanoparticles
1.2.2 Effects of the Adsorption of (Macro)Molecules
1.2.3 Effects of the Corrosion and Degradation of Nanoparticles
1.3 Physicochemical Modifications of Inorganic Nanoparticles Determine Their Biodistribution and Fate
1.3.1 Biodistribution: Nanoparticles Entering the Body
1.3.2 Subcellular Localization: Nanoparticles Entering the Cells
1.3.3 Long-Term Effects
1.4 Outlook and Conclusion
References
2 Biodistribution, Excretion, and Toxicity of Nanoparticles
2.1 Introduction
2.2 Biodistribution
2.2.1 Effect of Surface Material
2.2.1.1 PEGylation
2.2.1.2 Charge
2.2.2 Effect of Nanoparticle Size
2.2.3 Effect of Nanoparticle Shape
2.2.4 Effect of Nanoparticle Rigidity
2.2.5 Effect of Administration Route
2.3 Excretion
2.3.1 Mononuclear Phagocytic System Clearance
2.3.2 Renal Clearance
2.4 Toxicity
2.4.1 Liver Toxicity
2.4.2 Kidney Toxicity
2.4.3 Heart Toxicity
2.4.4 Brain Toxicity
2.4.5 Lung Toxicity
2.5 Challenges and Perspectives
References
3 Nanoparticle Interaction With Immune Cells for Nanoparticle-Mediated (Anticancer) Immunotherapy
3.1 Introduction
3.1.1 Nanoparticles
3.1.2 Innate Immune System
3.1.3 Adaptive Immune System
3.1.4 Immunotherapy
3.1.5 Nanoparticles as Anticancer Drug-Delivery System
3.2 Nanoparticles as Immunotherapy
3.2.1 Complementing Nanoparticle-Based Therapies
3.3 Nanoparticles as Vaccines Against Cancer
3.4 Nanoparticles as Diagnostics
3.5 Challenges
3.5.1 Modulating Innate and Adaptive Immunity
3.5.2 Nanoparticle Characteristics
3.6 Concluding Remarks
References
4 Clinical Translation of Nanomaterials
4.1 Diagnostics
4.1.1 Carbon Nanotubes
4.1.2 Nanoparticles
4.1.3 Quantum Dots
4.2 Therapeutics
4.2.1 Polymerics
4.2.2 Liposomes
4.3 Tissue Engineering
4.3.1 Scaffolds
4.3.2 Hydrogels
4.4 Conclusion and Perspective
Acknowledgment
References
5 Synthetic Receptors With Bioaffinity for Biomedical Applications
5.1 Introduction
5.2 Synthetic Receptors for Biomedicines
5.2.1 Toxin Neutralization
5.2.2 Bacteria Inhibition
5.2.3 Biomedical Imaging
5.2.4 Cancer Therapy
5.2.5 Cell Isolation
5.2.6 Other Potentials
5.3 Conclusion and Perspective
Acknowledgments
References
Part II: Biomedical Applications of Theranostic Bionanomaterials
Section I: Drug Delivery and Tissue Engineering
6 Calcium Phosphate Nanoparticle-Based Systems for Therapeutic Delivery
6.1 Introduction
6.2 Therapeutics Delivered Using Calcium Phosphate Nanoparticles
6.2.1 Type of Therapeutics
6.2.2 Intracellular Uptake and Release Mechanism
6.2.3 Factors Affecting Drug Release Rates
6.3 Types of Calcium Phosphate Nanoparticles
6.3.1 Bare Calcium Phosphate Nanoparticles
6.3.1.1 Core
6.3.1.2 Core–Shell
6.3.1.3 Multilayer
6.3.2 Coated Calcium Phosphate Nanoparticles
6.3.2.1 Lipid-Coated Calcium Phosphate Nanoparticles
6.3.2.2 Polymer Coating
6.4 Conclusion and Perspectives
Acknowledgments
References
7 Graphene and Graphene Oxide for Tissue Engineering and Regeneration
7.1 Introduction
7.2 Properties and Applications in Tissue Engineering
7.2.1 Mechanical Properties and Applications
7.2.2 Electrical Properties and Applications
7.2.3 Chemical Properties and Applications
7.2.4 Other Properties and Applications
7.3 Conclusion
References
8 Nanomaterial Design and Tests for Neural Tissue Engineering
8.1 Design of Nanomaterials for Neural Tissue Engineering
8.2 Nanomaterials for the Repair of Spinal Cord Injury
8.2.1 Design of the Nanomaterials for Repairing the Spinal Cord Injury
8.2.2 Nanomaterials Combined With Growth Factors for Repairing Spinal Cord Injury
8.2.3 Nanomaterials Combined With Cell Therapy for Repairing Spinal Cord Injury
8.3 Nanomaterials for the Repair of Peripheral Nerve Injury
8.3.1 Design of the Nerve Conduits for Repairing the Peripheral Nerve Injury
8.3.2 Nanomaterials Combined With Growth Factors for Repairing Peripheral Nerve Injury
8.3.3 Nanomaterials Combined With Cell Therapy for Repairing Peripheral Nerve Injury
8.4 Conclusions
Acknowledgments
References
Further Reading
9 Nanomaterials for Wound Healing: Scope and Advances
9.1 Introduction
9.2 Brief Introduction to Bionanomaterials on Wound Healing
9.3 Characteristics of Wound Healing: Normal and Abnormal
9.4 The Mechanism and Advantages of Bionanomaterials in Wound Healing
9.4.1 Antibacterial and Antiinflammatory
9.4.2 Bionanomaterials Can Promote Wound Healing Due to Extracellular Matrix Regulation Directly
9.4.3 Bionanomaterials Can Support Skin Regeneration by Promoting Stem Cell Growth
9.4.4 Bionanomaterials Can Modulate Growth Factors in the Wound Site
9.5 Potential Scope of Bionanomaterials in Wound Healing in Clinical Practice
9.5.1 The Application on Skin Wound Healing
9.5.1.1 Application of Bionanomaterials in Dressings
9.5.1.2 Application of Bionanomaterials in Suture Fabrication
9.5.1.3 Use of Bionanomaterials and Keloid
9.5.2 Bionanomaterials in Promoting Bone Fracture and Tendon Healing
9.5.3 Bionanomaterials in Promoting Neuron Repair
9.5.4 The Bionanomaterials Promoting Healing in the Abdomen
9.6 Obstacles to Bionanomaterial Application
9.7 Outlook of Bionanomaterials for Wound Healing in the Future
References
10 Advanced Nanovaccines for Immunotherapy Applications: From Concept to Animal Tests
10.1 Introduction
10.1.1 Immunotherapy
10.1.2 Nanotechnology for Immunotherapy
10.2 Design of the Systems
10.2.1 Size
10.2.2 Shape
10.2.3 Charge
10.2.4 Flexibility/Elastic Modulus
10.2.5 Surface Chemistry and Roughness
10.2.6 Adjuvants
10.2.7 Position of the Antigens
10.3 In Vitro Assessment
10.3.1 Murine Cells
10.3.2 Human Cells
10.4 In Vivo Models and Efficacy
10.4.1 Immunostimulation—Cancer
10.4.2 Immunomodulation—Rheumatoid Arthritis, Experimental Autoimmune Encephalomyelitis, Diabetes
10.5 Conclusions
Acknowledgments
References
Section II: Bionanomaterials for Diagnostics
11 Two-Dimensional Nanomaterials in Cancer Theranostics
11.1 Introduction
11.2 Theranostic Payloads
11.2.1 Biomedical Imaging
11.2.1.1 Optical Imaging
11.2.1.2 Magnetic Resonance Imaging
11.2.1.3 X-Ray Computed Tomography Imaging
11.2.1.4 Positron Emission Tomography Imaging
11.2.1.5 Photoacoustic Imaging
11.2.1.6 Others
11.2.2 Therapeutics
11.2.2.1 Chemotherapy
11.2.2.2 Photothermal Therapy
11.2.2.3 Photodynamic Therapy
11.2.2.4 Other Therapies
11.3 Theranostic Two-Dimensional Nanomaterials
11.3.1 Graphene and Its Derivatives
11.3.1.1 Gene and Drug Delivery
11.3.1.2 Graphene as a Phototherapeutic Agent
11.3.2 Transition Metal Dichalcogenides
11.3.2.1 Transition Metal Dichalcogenides as Imaging Agents
11.3.2.2 Transition Metal Dichalcogenides for Gene and Drug Delivery
11.3.2.3 Transition Metal Dichalcogenides as Photothermal Agents
11.3.3 Metal-Organic Frameworks
11.3.4 Graphitic Carbon Nitride
11.3.5 Black Phosphorus
11.3.6 Other Two-Dimensional Nanomaterials
11.4 Summary and Future Perspectives
References
12 Polymeric Micelles for Tumor Theranostics
12.1 Introduction
12.2 Polymeric Micelles for Tumor Imaging
12.2.1 Polymeric Micelles for Optical Imaging
12.2.2 Polymeric Micelles for Tumor Magnetic Resonance Imaging
12.2.3 Polymeric Micelles for Tumor Multimodality Imaging
12.2.4 Polymeric Micelles for Tumor Theranostics
12.3 Conclusions and Perspective
Acknowledgments
References
13 Theranostic Biomaterials for Regulation of the Blood–Brain Barrier
Abbreviations
13.1 Introduction
13.2 The Blood–Brain Barrier
13.2.1 The Blood–Brain Barrier’s Structure and Function
13.2.2 Role of the Blood–Brain Barrier in Central Nervous System Diseases
13.3 Explaining Theranostic Agents—A Growing Concept
13.4 Nanobiomaterials Used to Repair and/or Regenerate the Blood–Brain Barrier
13.4.1 Scaffolds
13.4.2 Carbon Nanotubes
13.5 Nanobiomaterials Used as Imaging and Diagnosing Agents at the Blood–Brain Barrier
13.5.1 Gold Nanoparticles
13.5.2 Quantum Dots
13.5.3 Superparamagnetic Iron–Oxide Nanoparticles
13.6 Conclusion and Future Perspectives
Acknowledgments
References
14 Upconversion Nanomaterials for Near-infrared Light-Mediated Theranostics
14.1 Introduction
14.2 Upconversion Nanoparticle Design Considerations
14.2.1 Luminescence Mechanism of Upconversion Nanoparticles
14.2.2 Synthesis and Surface Engineering
14.2.3 Upconversion Luminescence Tuning
14.3 Upconversion Nanoprobes for Biosensing
14.3.1 In Vitro Assays of Biomarkers
14.3.2 In Vivo Detection of Biomolecules
14.4 In Vivo Bioimaging Using Upconversion Nanoparticles
14.4.1 Near-Infrared Light-based Optical Imaging
14.4.2 Upconversion Nanoparticles for Multimodel Bioimaging
14.5 Photon Upconversion-Mediated Medical Therapy
14.5.1 Near-Infrared Light-Triggered Drug Delivery
14.5.2 Near-Infrared Light-Activated Photodynamic Therapy
14.5.3 Near-Infrared Light-Mediated Optogenetic Therapy
14.6 Toxicity Studies of Upconversion Nanoparticles
14.7 Conclusion and Outlook
References
15 Biofunctional Magnetic Nanomaterials for Diagnosis, Therapy, and Theranostic Applications
15.1 Introduction
15.2 Synthesis and Modification of Biofunctional Magnetic Nanomaterials
15.2.1 Synthesis of Magnetic Nanomaterials
15.2.2 Hybridization of Magnetic Nanoparticles with Different Morphologies
15.3 Magnetic Resonance Imaging-Based Multimodal Diagnosis
15.3.1 Magnetic Resonance Imaging–Computed Tomography Bimodal Diagnosis
15.3.2 Magnetic Resonance Imaging–ECT Bimodal Diagnosis
15.3.3 Magnetic Resonance Imaging-Based Multimodal Diagnosis
15.4 Magnetic Nanoparticles for Hyperthermia-Based Therapy
15.4.1 Magnetic Hyperthermia Therapy
15.4.2 Photothermal Therapy
15.4.3 Combined Therapy
15.5 Magnetic Nanoparticles for Theranostic Treatment
15.6 Challenges and Conclusions
Acknowledgments
References
Section III: Bionanomaterials for Biosensing and bioimaging
16 AIEgen-Based Fluorescent Nanoparticles for Long-Term Cell Tracing
16.1 Introduction
16.2 Fabrication of AIEgen-Based Nanoparticles
16.2.1 AIEgens
16.2.2 Introduce AIEgens Into Nanoparticles
16.2.2.1 Noncovalent Binding Method
16.2.2.2 Covalent Binding Method
16.2.3 Functionalization of the AIEgen-Based Nanoparticles
16.2.3.1 Enhancing Targeting Efficiency
16.2.3.2 Enabling Multifunctionality
16.3 Long-Term Cell Tracing With AIEgen-Based Fluorescent Nanoparticles
16.3.1 In Vitro Cell Tracing
16.3.2 In Vivo Long-Term Cell Tracing
16.4 Conclusions and Perspectives
References
17 Multimodal Carbon Dots as Biosensors
17.1 Introduction
17.2 Classification of Carbon Dots and Origins of Photoluminescence
17.2.1 Classification and Nomenclature of Carbon Dots
17.2.2 Origins of Photoluminescence of Carbon Dots
17.2.2.1 Quantum Confinement Effect and Collective Exciton Effect
17.2.2.2 Surface/Edge State of Graphene Quantum Dots
17.3 Synthesis of Carbon Dots
17.3.1 “Top-Down” Approaches
17.3.1.1 Laser Ablation
17.3.1.2 Arc Discharge
17.3.1.3 Electrochemical Carbonization
17.3.2 “Bottom-Up” Approaches
17.3.2.1 Pyrolysis—Solvothermal and Hydrothermal Carbonization
17.3.2.2 Microwave/Ultrasonic-Assisted Method
17.3.2.3 Template-Supported Method
17.4 Spectroscopic Properties of Carbon Dots
17.4.1 UV Absorption
17.4.2 Photoluminescence
17.4.3 Upconversion Photoluminescence
17.4.4 Phosphorescence
17.5 Biomedical Applications of Carbon Dots
17.5.1 Biocompatibility and Bioimaging of Carbon Dots
17.5.2 Carbon Dots as Biosensors
17.5.3 Theranostic Carbon Dots
17.6 Conclusion
References
18 Bionanomaterials as Imaging Contrast Agents
18.1 Introduction
18.2 Magnetic Contrast-Enhancing Bionanomaterials
18.2.1 Magnetic Resonance Imaging
18.2.2 Magnetic Particle Imaging
18.2.3 Magnetomotive Imaging
18.3 Optical Contrast-Enhancing Bionanomaterials
18.3.1 Luminescence
18.3.2 Fluorescence Resonance Energy Transfer
18.3.3 Raman
18.3.4 Optical Coherence Tomography
18.3.5 Photoacoustic Imaging
18.4 Acoustic Contrast-Enhancing Bionanomaterials
18.5 X-Ray Contrast-Enhancing Bionanomaterials
18.6 Conclusions
References
19 Nucleotide Aptamers as Theranostic Biomaterials
19.1 Introduction
19.2 The Structure of Aptamers and Complexes
19.2.1 Primary Aptamer Structures
19.2.2 Cocrystal Structures of Aptamers With Ligands
19.2.3 Aptamer Structure Prediction
19.3 Applications of Aptamers
19.3.1 Therapeutical Nucleotide Aptamers
19.3.1.1 Aptamers for Cancer Therapies
AS1411
NOX-A12
19.3.1.2 Aptamers Against Age-Related Macular Degeneration
Macugen
19.3.1.3 Aptamers for Antithrombotic Therapy
19.3.1.4 Aptamers Against Other Diseases
19.3.2 Aptamer–Drug Conjugates for Targeted Therapies
19.3.2.1 Aptamer–Chemotherapeutic Conjugates
19.3.2.2 Targeted Drug-Delivery Vehicles Conjugated With Aptamers
19.3.3 Biosensors Made From Aptamers
19.3.3.1 Electrochemical Detection
19.3.3.2 Optical Detection
19.3.3.3 Other Detection Methods
19.3.4 Diagnostic Applications
19.4 Conclusion and Future Perspective
Acknowledgments
References
20 Electronic Structures of Alkaline Rare Earth Fluoride-Based Upconversion Nanomaterials
20.1 Introduction
20.2 Calculation Setup
20.3 Results and Discussions
20.4 β-NaYF4
20.5 β-NaGdF4
20.6 β-NaLuF4
20.7 Summary
Acknowledgments
References
21 Lanthanide-Based Magnetic Resonance Imaging Metal-Responsive Agent Overview
21.1 Introduction
21.2 Gadolinium Magnetic Resonance Imaging-Responsive Agents
21.2.1 The Relaxivity Change Mechanism
21.2.2 Survey of Gd-Based Magnetic Resonance Imaging Sensors
21.2.2.1 Modulation of the Inner Sphere Number of Water Molecules
21.2.2.2 Modulation of Rotational Tumbling Time
21.3 Survey of the Paramagnetic Chemical Exchange Saturation Transfer Responsive Agent
21.3.1 Zinc and Calcium Chemical Exchange Saturation Transfer Responsive Agent
21.4 Conclusions and Future Perspectives
References
Index
Back Cover

Citation preview

Theranostic Bionanomaterials

Theranostic Bionanomaterials Edited by

Wenguo Cui Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P.R. China

Xin Zhao Department of Biomedical Engineering, The Hong Kong Polytechnic University, Hong Kong SAR, P.R. China

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2019 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-815341-3 For Information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Matthew Deans Acquisition Editor: Sabrina Webber Editorial Project Manager: John Leonard Production Project Manager: Debasish Ghosh Cover Designer: Greg Harris Typeset by MPS Limited, Chennai, India

List of Contributors Andreia Almeida

i3S—Institute of Investigation and Innovation in Health, Univerisity of Porto, Porto, Portugal; INEB—National Institute of Biomedical Engineering, University of Porto, Porto, Portugal; ICBAS—Abel Salazar's Institute of Biomedical Sciences, University of Porto, Porto, Portugal

Ho Pan Bei Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, P.R. China

Xia Cao

Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, United States; Department of Pharmaceutics and Tissue Engineering, School of Pharmacy, Jiangsu University, Zhenjiang, P.R. China

Eudald Casals

School of Biotechnology and Health Sciences, Wuyi University,

Jiangmen, China

Gregori Casals

Department of Biochemistry and Molecular Genetics, Hospital Clinic, IDIBAPS, Barcelona, Spain

Chaenyung Cha

School of Materials Science and Engineering, Ulsan National Institute of Science and Technology, Ulsan, South Korea

Qiushui Chen Department of Chemistry, National University of Singapore, Singapore, Singapore Sijie Chen

Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong, P.R. China

Wei Chen

Institute for Advanced Materials, School of Materials Science and Engineering, Jiangsu University, Zhenjiang, P.R. China; College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao, P.R. China

Wenguo Cui

Shanghai Institute of Traumatology and Orthopaedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P.R. China

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List of Contributors

Lianfu Deng

Shanghai Institute of Traumatology and Orthopaedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P.R. China

Juan Du Department of Surgery, The University of Hong Kong, Hong Kong SAR, China; Department of Microbiology, Tumor and Cell Biology, Centre for Translational Microbiome Research (CTMR), Visionsgatan 4, Karolinska Institutet, S-17164 Stockholm, Sweden Patrícia Figueiredo

Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland

Flavia Fontana Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland Hui Gao Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong, P.R. China Jisu Hong

School of Materials Science and Engineering, Ulsan National Institute of Science and Technology, Ulsan, South Korea

Wei Hu Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, P.R. China

Bolong Huang

Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, P. R. China

Di Huang Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, United States; Department of Biomedical Engineering, Research Center for Nano-biomaterials and Regenerative Medicine, College of Biomedical Engineering, Taiyuan University of Technology, Taiyuan, P.R. China Per Hydbring Department of Oncology and Pathology, Visionsgatan 4, Karolinska Institutet, S-17164 Stockholm, Sweden

Mirae Kim School of Materials Science and Engineering, Ulsan National Institute of Science and Technology, Ulsan, South Korea Yiwei Li

Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States

List of Contributors

xv

Caihou Lin

Department of Neurosurgery, Fujian Medical University Union Hospital, Fuzhou, P.R. China

Han Liu Shanghai Institute of Traumatology and Orthopaedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P.R. China; Department of Nanotechnology Engineering, University of Waterloo, Waterloo, ON, Canada

Kai Liu State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, P.R. China

Yongjian Liu

Mallinckrodt Institute of Radiology, Washington University, St. Louis, MO, United States

Haiwei Lu John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, United States; State Key Laboratory for Manufacturing Systems Engineering, Xi’an Jiaotong University, Xi’an, P.R. China Yue Ma

School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, P.R. China

Peng Mi

Department of Radiology, Center for Medical Imaging, and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Collaborative Innovation Center for Biotherapy, Chengdu, P.R. China

Rui Pedro Moura

CESPU—Institute of Investigation and Advanced Formation in Health Sciences and Technologies, Gandra, Portugal

Guoqing Pan

Institute for Advanced Materials, School of Materials Science and Engineering, Jiangsu University, Zhenjiang, P.R. China

Yue Pan

Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, P.R. China

Hao Pei

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, United States

Yun Piao Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, P.R. China

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Soraia Pinto i3S—Institute of Investigation and Innovation in Health, Univerisity of Porto, Porto, Portugal; INEB—National Institute of Biomedical Engineering, University of Porto, Porto, Portugal

Dhayakumar Rajan Prakash

Department of Pharmaceutical Science Laboratory, Åbo Akademi University, Turku, Finland

Victor Puntes

Vall d’Hebron Institut of Research (VHIR), Barcelona, Spain; Catalan Institute of Nanoscience and Nanotechnology - ICN2 (BIST - CSIC), Campus de la UAB, 08193 Bellaterra, Barcelona and Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg. Lluis Companys 23, 08010 Barcelona, Spain

Yaping Qi

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, United States; Department of Physics, The University of Hong Kong, Pokfulam, Hong Kong

Zheyan Qian

Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, P.R. China

Liangliang Qu John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, United States

Jessica M. Rosenholm

Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland

Hélder A. Santos Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland Bruno Sarmento CESPU—Institute of Investigation and Advanced Formation in Health Sciences and Technologies, Gandra, Portugal; i3S—Institute of Investigation and Innovation in Health, Univerisity of Porto, Porto, Portugal; INEB—National Institute of Biomedical Engineering, University of Porto, Porto, Portugal Luoran Shang

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, United States

Flávia Sousa CESPU—Institute of Investigation and Advanced Formation in Health Sciences and Technologies, Gandra, Portugal; i3S—Institute of Investigation and Innovation in Health, Univerisity of Porto, Porto, Portugal; INEB—National Institute of Biomedical Engineering, University of Porto, Porto, Portugal; ICBAS—Abel Salazar's Institute of Biomedical Sciences, University of Porto, Porto, Portugal

List of Contributors

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Deborah Sultan Mallinckrodt Institute of Radiology, Washington University, St. Louis, MO, United States Allison Tam

Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, P.R. China

Peter van Nooten

Shanghai Institute of Traumatology and Orthopaedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P.R. China; Department of Nanotechnology Engineering, University of Waterloo, Waterloo, ON, Canada

Jiuhai Wang

Department of Biomedical Engineering, The Hong Kong Polytechnic University, Hong Kong S.A.R., P.R. China

Kenneth K.Y. Wong

Department of Surgery, The University of Hong Kong,

Hong Kong SAR, China

Zhengwei Wu

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, United States; Department of Biomedical Engineering and Biotechnology, University of Massachusetts Lowell, Lowell, MA, United States

Lili Xie

College of Chemistry, Fuzhou University, Fuzhou, P.R. China

Jiajia Xue The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, United States

Huang-Hao Yang

College of Chemistry, Fuzhou University, Fuzhou, P.R.

China

Mo Yang

Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, P.R. China

Yuhe Yang

Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, P.R. China

Hongbo Zhang

Department of Pharmaceutical Science Laboratory, Åbo Akademi University, Turku, Finland; Turku Center for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland

Huaping Zhang

Department of Radiology, Center for Medical Imaging, and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Collaborative Innovation Center for Biotherapy, Chengdu, P.R. China

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List of Contributors

Lei Zhang

State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, P.R. China

Qiang Zhang

Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, P.R. China

Weixia Zhang

John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, United States

Yu Shrike Zhang

Division of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, United States

Yuezhou Zhang

Department of Pharmaceutical Science Laboratory, Åbo Akademi University, Turku, Finland

Xin Zhao

Department of Biomedical Engineering, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, P.R. China

Yongfeng Zhao MS, United States

Department of Chemistry, Jackson State University, Jackson,

About the Editors Wenguo Cui is a full Professor in the Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Dr. Cui holds a PhD in Biomaterials and his research expertise is in nanotechnology for tissue regeneration, drug delivery, and nanomedicine.

WENGUO CUI

Xin Zhao is an Assistant Professor in the Department of Biomedical Engineering, The Hong Kong Polytechnic University, Hung Hom, Hong Kong. Dr. Zhao holds a PhD in Biomaterials and Tissue Engineering from University College London and her research interests include biomaterials, tissue engineering, drug delivery, cell microenvironment, and microfluidics.

XIN ZHAO

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1 Biodistribution, Excretion, and Toxicity of Inorganic Nanoparticles Eudald Casals1, Gregori Casals2, Victor Puntes3, Jessica M. Rosenholm4 1

SCHO OL OF BIOTECHNOLOGY AND HE ALTH SC IENCES, WUYI UNIVERSITY, JIANGMEN, C HI N A 2 DEPARTME NT OF BIOCHEMISTRY AND MOLECULAR GENETICS, HOSPITAL CLINIC, IDIBAPS, BARCELONA, SPAIN 3 VALL D ’HEBRON INSTITUT O F R ESEARCH (VHIR), BARCELONA, SPAIN; CATALAN INSTITUTE OF NANOSCIENCE AND NANOTECHNOLOGY - ICN2 (BIST - C SIC), CAMP US DE LA UAB, 08193 BE LLATERRA, BARCELONA AND INSTITUCIÓ CATALANA DE RECERCA I E STUDIS A VANÇAT S (ICREA), PG. LLUIS COMP ANYS 23, 08010 BARCELONA, SPAIN 4 P HA R MA C E UT I C AL S C I E NC E S L ABOR AT ORY , FACUL TY O F S C I ENC E AND ENGI NE ER I NG, ÅB O A K A DE MI U N I V E RS I T Y , T U RK U , F I N L AND

Abbreviations CNTs NIR NPs QDs SPR UCNPs

carbon nanotubes near-infrared region nanoparticles quantum dots surface plasmon resonance upconverting nanoparticles

1.1 Introduction: Inorganic Nanoparticles and Their Interest in Medicine With the advent of more increasingly complex nanostructures ensuing from the high demands posed by the requirement for more personalized treatments and precision medications, inorganic nanomaterials have emerged as flexible platforms for the development of theranostic nanomedicines. Owing to their inherent detectability by many biomedical imaging techniques combined with the robust inorganic structures providing shelter and/or stability to incorporated active molecules, some even exhibiting intrinsic therapeutic action (e.g., phototherapy, antiinflammatory), inorganic nanomaterials are perfectly suited for performing combined diagnostics and therapy [1]. In addition, inorganic nanomaterials and nanoparticles (NPs) represent a vast array of different materials, including, for instance, metals and metal oxides, nonoxide ceramics, semiconductor nanocrystals [quantum dots (QDs)], magnetic NPs, upconverting phosphors (UCPs), or upconverting NPs (UCNPs) and Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00001-8 © 2019 Elsevier Inc. All rights reserved.

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carbon nanostructures (e.g., nanotubes, fullerenes, carbon dots, graphene, nanodiamonds) that can, in a single platform, perform the different activities mentioned [2]. These advantages have spurred the substantial growth of studies exploring new possibilities for medicine. Thus, among the pool of newly proposed materials, synthesized inorganic nanomaterials are being increasingly studied as alternative tools in medical applications. The main advantages of these materials include their successful use as robust drug carriers, as antennas that can be excited in biologically transparent environments, and being able to adjust the activity of conjugated biomolecules. Furthermore, their unique physicochemical signatures allow their tracking and easy detection in biological environments. All this is being translated into a new generation of diagnostics, imaging agents, and therapies for detecting and treating disease in its earliest stages [3]. Moreover, their interest is expanded toward the possibility of combining these advances to enable the creation of multimodal/multifunctional nanosized particles that may combine diagnosis together with different therapies (chemo-, thermo-, radio-, immunotherapies, and so on) with synergistic effects, superior to any currently used treatment [1,4]. This is especially important, for instance, for cancer treatment, since survival rates critically depend on the stage that cancer is diagnosed and single-modality treatments usually cannot overcome the associated drug resistance. Thus, the advantages of inorganic materials to create advanced functional NP-based platforms for detection and multimodal treatments of different diseases make them ideal candidates to be used as tools for the future of medical progress (Fig. 1 1). Some of the main advantages of inorganic NPs for medicine can be summarized as follows: 1. Size considerations: possibilities for drug delivery. Inorganic NPs are small, and can therefore interact with molecular biological structures in a unique manner. Of course, NP

FIGURE 1–1 Overview of the versatility of inorganic nanoplatforms for medical applications.

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size control is a paradigmatic feature for their claimed potential in nanomedicine. Size influences key biological interactions such as association with proteins, biodistribution, and clearance rates. It is accepted that the final fate of NPs is strongly influenced by their size. For stable, low-interacting and nonimmunogenic NPs, it is considered that the smallest ones (,6 nm, core 1 surfactant) are rapidly cleared by the kidneys and the largest ones (.100 nm) are also easily removed from blood circulation by the cells of the mononuclear phagocyte system [5] (Fig. 1 2). The variety of sizes and the narrow size distributions that can nowadays be easily produced [6] has enabled a better understanding of the role played by this property in parameters that are important for different medical applications, such as accumulation and penetration in targeted organs or tumors [7]. Importantly, their small size, similar to those of proteins, allow long lifetimes in blood, a better use of the enhanced permeation and retention effect observed in solid tumors and in atherosclerosis, which is associated with chronic inflammation of arterial blood vessels [8], and an increased tissue or tumor penetration (see, for example, Barua et al. [9]). Here, it is widely accepted that a compromise between accumulation and penetration of drugs in the targeted area has to be reached. For instance, in cancer treatment, a cocktail of NP sizes can strike the tumor in different areas: larger NPs are more readily accumulated in the tumor, but they are restricted to regions on the periphery, close to blood capillaries, while smaller NPs are able to penetrate deeper into tumors [1,9]. Therefore, due to the NP morphology control that the scientific community is achieving, inorganic NPs are being widely studied as drug-delivery devices. They can be used as efficient drug carriers, allowing a high dose of drug to arrive at more delayed and intermittent times to specific targets, while protecting the drug and promoting endocytosis [10]. Also, they can modify the biodistribution of the drug in the body, tissues, and cells [11]. Remarkably, here, the versatility in the preparation of different inorganic NPs allows the

FIGURE 1–2 The size of NPs determines the interaction with the immune system and hence the residence time in blood and associated effects. NP, Nanoparticles.

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possibility of modifying pharmacokinetic aspects such as drug solubility, systemic distribution, metabolism, and elimination. All these are important factors that not only can increase the amount of drug reaching the targeted area but also lower the toxicity to normal tissues [12]. Spherical NPs are usually the chosen option for use as carriers due to their simple synthesis and easy functionalization. However, exotic shapes, such as nanorods, nanostars, nanocages, and nanoshells, among others, can be finely synthesized in research laboratories and have also been proposed for biomedical applications, especially in imaging but also for therapy [13]. For example, the extravasation rate of rod-like NPs was reported to be higher than that for equivalent spheres due to tumbling effects [14] and cellular uptake and bacterial killing efficiencies have been reported to be higher for rod-shaped silica NPs as opposed to their spherical counterparts [15]. 2. Surface considerations: possibilities enabled by surface functionalization. Inorganic NPs can be tuned and adjusted with a proper and controlled functionalization with specific biomolecules. The modification of NP surface properties (functionalization with stealth agents, controlling surface charge) will enable to adjust on a case-by-case basis the desired behavior inside the body. Here, the most paradigmatic case is targeted therapies. Since the discovery of the trans-activating transcriptional activator from human immunodeficiency virus 1 that can be efficiently taken up from the surrounding media by different cell types in culture [16], numerous peptides and small molecules have been developed to target specific therapies to different cell types and subcellular structures. The flexibility of the NP surface for functionalization using different chemistries makes them ideal tools for these targeted therapies [17]. In addition, the possibilities of the rational control on the functionalization of inorganic NPs with biomolecules is particularly important for immunotherapy, the training of the immune system to attack specifically cancer cells, or to boost the immune system in a very general way, similarly to what vaccines can do [18]. In this regard, inorganic NPs are excellent antigen presenters, for example, monoclonal antibodies designed to recognize and attack a very specific target of tumoral cells [19] or well-tolerated adjuvants to enhance immunogenicity, as has been observed in different studies (see, for example, Ref. [20] and references therein). Again, the use of NP conjugates as adjuvants may present some natural advantages: rational design, low toxicity, low cost, and modified and modifiable biodistribution. Furthermore, packing molecules onto NP surfaces to protect them against degradation has been known about for decades [21] and the protection of the surface of the nanostructures with biological molecules such as albumin can improve their biocompatibility [22]. This is the case of Abraxane, considered the first formulation of chemotherapeutic drugs combined with the NP albumin bound (nab) technology to deliver and reduce side effects of paclitaxel for the treatment of different types of cancer [23]. 3. Inorganic NPs can also be used as therapeutic agents by themselves. First, it is possible because inorganic NPs can interact with photons of different wavelengths and trigger a variety of physical processes [12a]. Probably, the case that has attracted most attention in the scientific community is surface plasmon resonance (SPR) in the near-infrared (NIR) region

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of metallic NPs [24]. The region of the spectra in which this SPR absorbs is strongly dependent on the shape of the NPs, and they can be designed to absorb at specific desired wavelengths. This is commonly achieved by using anisotropic NPs (e.g., Au nanorods [25], nanostars [26], and nanocages [27]). NIR is a region of the light spectrum where there is the window of optical transparency (also known as the therapeutic window). Simplifying, it is a window of transparency for biological tissues from the overlapping light absorption of water, hemoglobin, and melanin, basically. Thus the possibility to excite in the NIR region rather than ultraviolet radiation allows for both minimization of photodamage of biological specimens and maximization of the penetration depth into the tissue of the excitation light. Beyond imaging, an interesting application of inorganic NPs is in photothermal therapy, where NIR light absorbed by NPs can increase the temperature up to levels for cellular death in the vicinity of the NPs, but not in unlabeled tissue [12a,28]. At those wavelengths, photons can penetrate deep into tissue, enabling the tumors to be reached. Here, UCNPs, which exhibit photon upconversion (two or more incident photons within the NIR region are absorbed by the UCNPs and converted into one emitted photon with higher energy [29]) are especially suited as well to combine both molecular imaging and selective photothermal therapy [30]. Other examples of the use of NPs as therapy per se are the use of magnetic NPs to treat cancer by inducing hyperthermia [1,31], such as the FDA-approved Nanotherm (based on superparamagnetic iron oxide NPs), and the antioxidant activity of CeO2 NPs to treat conditions related to oxidative stress and chronic inflammation [32]. 4. Inorganic NPs possess unique physicochemical properties, different from cells and tissues: there are possibilities for tracking their evolution and biodistribution. Here, it is worth mentioning that research and clinics have a much broader tradition in using organic NPs and they arrived in the clinic before inorganic NPs, as in the case of Doxil, a liposomal formulation (hundreds of nanometers in size, biocompatible, and biodegradable) of doxorubicin that increases the solubility of the active ingredient and modifies the dosage by sustaining it over time [33]. However, although organic NPs are generally simple to make and normally readily biodegradable, they are difficult to characterize and it is hard to monitor and trace their evolution and biodistribution inside the body. In contrast, the unique physicochemical different signatures of inorganic NPs allow their easier detection in biological media and more accurate monitoring of their evolution and distribution in physiological environments and the body [3a]. To this, many inorganic materials possess intrinsic properties that can be exploited for different advanced imaging techniques, including superresolution microscopy (e.g., nanodiamonds [34]), two-, three-, or multiphoton microscopy (e.g., UCNPs [35], ZnO NPs [36], and other nonlinear optical techniques [37]). Being electron-dense materials, inherently photoluminescent inorganic NPs may further be suitable for correlative light and electron microscopy [38], rendering them suitable as intracellular dual-contrast markers for studying intracellular processes and trafficking of biomolecules. Owing to the robust inorganic matrix, incorporated molecular imaging agents can also be readily photostabilized for long term in vivo imaging applications not attainable by the molecular imaging agent itself [39].

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Despite all these interesting advantages and promising results obtained in the research using nanomaterials for medicine, only a few of them have reached the bedside [40]. These mainly include liposomes or organic particles but also FeOx, Au, or SiO2 NPs are already approved, for example, by the American Food and Drug Administration for clinical trials. A review from 2016 identified 51 FDA-approved nanomedicines and around 77 products in clinical trials [41]. There are some shortcomings in the application of NPs in clinics that may be responsible for hindering a faster progress of nanomedicine. One of them, and similar to any drug development enterprise, is the enormous financial needs for new drugs and medical technologies to reach the market and patients. As economic aspects are out of the scope of this chapter, we focus on the gaps in the knowledge and technical aspects to take into account to increase the number of nanomaterial systems to reach clinical applications. A major one is that, still, the safety of nanomaterials is a subject of wide debate. Here, it is key to consider that the response of nanomaterials inside organisms or when released to the environment is complex and diverse, and a variety of parameters are involved. Nanomaterials may be unstable and agglomerate, yielding microscopic particles, or they may end up embedded in exposed materials. Indeed, this aggregation may entail toxic effects as the lung toxicity described in Section 1.2.1. From this same instability, nanomaterials may corrode and dissolve into molecular or ionic species, or they can suffer morphological modifications. Release of toxic ions may happen if any of the constructs are composed of such elements and, thus, NPs may act as a reservoir for them (see Section 1.2.3). Importantly, the surface of the nanomaterials, which determines their bioactivity, experiences constant modifications, particularly the adsorption of macromolecules from the media where they are exposed. This protein absorption in the bloodstream not only modifies NP surface properties but also may result in protein changes and alter their metabolization (see Section 1.2.2). Thus, in physiological environments and inside the body, biodistribution and the fate of nanomaterials will mainly depend on those parameters, modified by processes that, in turn, depend on the characteristics of the exposure media [42]. And moreover, the characterization of the evolution of NPs in complex matrices such as biological environments and inside the body (intracellular, tissue, and organ) is still a challenge and limited reliable data are available, adding more confusion and hampering nanomedicine development. A better knowledge of how they interact with cells, tissues, and organs is still required; including their subsequent release and/or intracellular degradation, transfer to other cells, and/or translocation across tissue barriers, the biodistribution and kinetics, and their modes of clearance. All this will be reviewed and discussed below.

1.2 Physicochemical Modifications of Inorganic Nanoparticles in Physiological Environments Determine Their Effects: Safety and Toxicity Considerations The safe and effective use of promising NP-based solutions for medical problems needs a proper evaluation and assessment of their behavior (ADME profiles) in the physiological

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media and possible unwanted effects. In this sense, despite the vast range of publications that addresses toxicity and safety aspects of nanomaterials, the potential benefits and risks that their use in medicine entails are still being debated. The poor knowledge of nanomaterials responses and evolution inside biological media is recognized as one of the key points underpinning these controversies [43]. A paradigmatic case is the use of CeO2 NPs in medicine. While it has been reported many times to be beneficial protecting against oxidative stress [32], other studies, mainly related to the toxicity of the CeO2 nanopowders employed in industry, show toxicity in vitro and in vivo [44]. In addition, while some studies show antiinflammatory effects and that CeO2 NPs are taken up by hepatocytes [32d,45], others report macrophage (Kupffer cells) uptake by the liver [46]. Similarly, in the case of iron oxide NPs (including superparamagnetic iron oxides NPs and ultrasmall superparamagnetic iron oxides), one of the first inorganic nanomaterial employed in medical research [47], reports of potential medical benefits and others about toxic effects have been simultaneously published. While some reports show promising nerve cell regeneration activity [48], others find toxicity to neuronal cells [49]. As stated, the modifications of nanomaterials in physiological media lie at the root of these controversies. Inorganic NPs employed in nanomedicine research are unstable once out of their synthesis media and inserted in physiological environments and in the bloodstream, thus partially modifying their as-synthesized characteristics. It is known that once NPs are produced, they tend to minimize the high surface energy following what is called the Gibbs Thomson effect. This effect refers to the observation that small crystals of a liquid melt at a lower temperature than the bulk. This is explained as being due to the fact that as the size decreases, the surface tension increases. That is why NPs are systems in a metastable phase, and to reduce the surface area exposed, their fate is aggregation or dissolution toward more stable phases [50]. The most significant alterations the NPs undergo that affect their biological fate and effects are depicted in Fig. 1 3 and can be summarized as: (1) the formation of the NP protein corona (PC) as a result of the adsorption of proteins onto the inorganic surface, (2) NP corrosion and/or dissolution into ionic species, and (3) the agglomeration and aggregation of NPs, since this determines their proper interaction with biological entities.

1.2.1 Effects of the Agglomeration and Aggregation of Nanoparticles It is known that the NP tendency to aggregate in biological fluids depends on parameters such as their surface charge or coating, as well as the characteristics of the medium in which they are dispersed (ionic strength, pH, presence of macromolecules, proteins, etc.). For example, Cho et al. [51] and Casals et al. [52] reported how NPs showed a dramatic change in their state of aggregation, dispersibility, and charge upon transfer from a buffered aqueous solution to commonly used cell culture media. When NPs are destabilized in biological media, the special physicochemical properties that arise at the nanoscale (quantum confinement, superparamagnetism, extreme catalytic activity, etc.) are progressively/partially lost. Neither the properties nor the dynamics remain the same. Agglomeration entails

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FIGURE 1–3 Scheme of the physicochemical modifications that NPs can undergo in biological environments. NP, Nanoparticles.

modifications in terms of specific surface area, concentrations, mobility, and so forth; which can be very different from those of the as-synthesized NPs [50]. Needless to say, this can modify NP behavior and effects. For instance, in in vivo conditions, highly agglomerated NPs will be less mobile than stable ones in the bloodstream, and thus, it can affect the NP concentration in different parts of the body, they can be accumulated or trapped in specific organs and/or they may not reach the targeted organ [53]. Following this reasoning, when assessing NP effects, their aggregation state has often been the source of misleading conclusions. Following the Paracelsus statement of five centuries ago, sola dosis facit venenum (only the dose makes the poison), an accurate determination of the dose—i.e. the number of NPs and their surface area being considered the dosing determining parameters [54]—is critical to properly assess the potential toxicity of a material. As a consequence, in some reports the onset of toxicity in viability experiments might be related to the onset of agglomeration: in vitro, NP agglomerates “rain” on top of cells due to the density, thus changing the object to test (no longer NPs) and increasing the dose on the cell [51,55]. These large particles are indeed more difficult to be processed by the cells, as in the cases of frustrated phagocytosis of long ( . 5 μm) carbon nanotubes (CNTs) that caused chronic inflammation in the study of Poland et al. [56], and intracellularly they could be a (too large) stone in the cell machinery (e.g., average mesh size enabled by cytoskeletal filaments is around 100 nm [57]).

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Thus even if NPs are not toxic by themselves, they may entail a potential risk as a source of aggregates when considerations about their colloidal stability have not been taken into account. In this context, an example that deserves special attention is NPs prepared as powders. These types of materials, often called nanopowders, are massively produced by industry and represent a broad range of materials to be used, especially in low-tech applications, from textile to cosmetics, but also sometimes in research. However, this type of material could also enter into contact with biological entities, for example, through the skin, by inhalation, or even accidental ingestion [53,54]. To assess the biological impact of these types of commercial nanomaterials, the dry material has to be dispersed in physiological buffers prior to their exposure to cells or animal models. Here, the choice of the appropriate resuspension protocol is key to understanding the obtained results.

1.2.2 Effects of the Adsorption of (Macro)Molecules In order to analyze the NP behavior in physiological media, it is key to understand how NP and biological systems relate to each other. NPs interact with their environment through the surface, which is subject to continuous changes, while living cells communicate to the exterior mainly through their membrane proteins. It is well known that among the building blocks of life, proteins are of fundamental importance. In addition to its structural properties, almost all the interactions by which a cell recognizes and relates to what is around it (recognition of signals to induce a response, immune recognition, etc.) are mediated by proteins. The two main characteristics that allow this interfacial role of proteins are their amphipathic character (combination of polar and nonpolar residues which enable proteins to have a three-dimensional structure and to be in contact with different environments) and the large number of hydrogen bonds and hydrophobic interactions that a single protein can perform [58]. Already, in the first half of the 20th century, when the adsorption of proteins to implants started to be a concern in the medical community, hydrophobicity and surface charge were identified as the main factors to take into account to explain protein adsorption to inorganic surfaces [58a]. The interfacial chemistry between blood serum proteins and inorganic surfaces is a dynamic process governed by the Vroman effect [59]. In 1962 Vroman reported how the exposure of hydrophobic inorganic powders to blood plasma resulted in the removal of coagulation factors, and the inorganic surface became more hydrophilic [60]. Further, he showed that protein adsorption follows a competitive hierarchy: the highest mobility proteins arrived first and were later replaced by less motile proteins that had a higher affinity for the surface, mainly factor V and fibrinogen, in a process that takes up to a few hours [59b]. This process is recognized as the general phenomenon governing the competitive adsorption of a complex mixture of proteins (as serum) to surfaces, as pointed out by Slack and Horbett [61]. Furthermore, since the 1950s, studies of other interfacial phenomena involving proteins have identified the adsorption of proteins to inorganic surfaces as a process that evolves to an irreversible state. Initially, the strongest argument for this irreversibility was that proteins are provided with multiple, although weak, anchor points.

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However, detailed studies suggest other mechanisms. The work of Alaeddine and Nygren [62] pointed out the possibility that protein distribution on surfaces follows a crowding process where once the first proteins are attached, an initial cluster of proteins forms around these, thereby stabilizing them, and this mechanism is repeated until the entire surface is filled. Thus, along with the affinity of the protein for the surface, other mechanisms have been identified as determining factors that make the adsorption definitive; such as molecular relaxation time or spreading, and mechanisms depending on the time that proteins remain on the surface [22,58a,63]. Accordingly, all these interfacial processes take place when NPs are dispersed in biological media, for example, when incubated with cells in in vitro studies or after intravenous (i.v.) injection. However, some specificities in the case of inorganic NPs must be considered: NPs are not a fixed substrate but they move in solution, they have similar dimensions to proteins, and they possess a high curvature radii, thus changing the accessibility to their inorganic surfaces. All these effects modify the kinetics of the encounter between the NP surface and proteins, the mechanisms of attachment, and the biological outcome [64]. Grouped under the name of PC formation, these processes are key to understanding NP behavior in biological systems. It is recognized that the proteins forming the “corona” remain associated with the particles under normal conditions of in vivo and in vitro exposure, thereby conferring their biological identity to the NP PC composite and determining the interactions between NPs and the host in living systems. In other words, this corona of proteins “expressed” at the surface of the particle is what is “read” by cells [65]. There are plenty of references about PC formation studies in the case of inorganic NPs, to name a few using metal NPs such as AuNPs [22,52,66] and AgNPs [22]; hybrid metallic (FePt) NPs [67]; metal oxide NPs such as SiO2 [65], Fe3O4, CoO, CeO2 [22], TiO2, and ZnO NPs [68]; CdSe QDs [69] and CdSe/ZnS QDs [67]; among many others. Many of these studies combine the mechanistic aspects of the corona formation together with their biological effects. Regarding biological implications, the association with proteins may indeed biocompatibilize foreign matter such as NPs, which could result in detoxification of problematic particles, as in the case of albuminization of drugs with severe side effects, as in the already-mentioned Abraxane case [23]. Also important here is that the PC determines the NP surface charge displayed in biological media. Surface charge has been recognized as a key parameter that strongly influences the approaching of NPs to negatively charged biological membranes, and therefore determines internalization, immune response, and toxicity. For decades it has been known how positively charged macromolecules display an incremented toxicity profile compared with their neutral and negative counterparts (see, for example, Refs. [70,71]). Inspired from this, several studies have been carried out involving NPs of the same composition arranged with different surface characteristics. Hoshino et al. [72] evaluated the toxicity of ZnS-coated CdSe QDs, modified with carboxylic acids (negative), polyalcohols (neutral), and amines (positive). The results showed that, consistent with the case of polymers, the more positively charged the NPs, the higher the cytotoxicity. In another study, Goodman et al. [73] found similar results using 2 nm core AuNPs with different surface characteristics. In addition, surface charge did not only affect the interaction

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with cell membranes but also affected their biodistribution and residence time in the organism. Balogh et al. [74] encapsulated AuNPs of different sizes into dendrimers, providing negative and positive charges to the different composites. The results showed that the particles selectively accumulate in different organs depending on size or charge alone. In this report, for instance, comparing particles of the same size, the positive ones persisted in the kidneys for up to 4 days (and were finally excreted mostly in urine), whereas the negative and neutral particles remained in the liver and spleen over the analysis period. All in all, these results show that the effects of NPs in contact with biological systems have to be analyzed, together with the data about their PC formation process.

1.2.3 Effects of the Corrosion and Degradation of Nanoparticles The corrosion, dissolution, or disintegration processes of different materials have been widely studied for bulk materials in different areas of human activities, such as the iron corrosion (rust) associated with degradation of iron-based tools and structures like bridges. Potential risks and hazards derived from the introduction of metal ions in biological environments and ecosystems have also been studied for a long time. The ability of metallic particles to release metal ions and their induced toxicity has been the focus of many safety studies, as happens with any metallic implant where wear corrosion greatly contribute to the release of ions responsible for health-related problems [75]. Importantly, in biological environments, ions released may end up as different defined chemical species (speciation), and the different chemical species may induce different biological impacts [76]. Inorganic NPs are also subjected to these processes. Due to their reduced size, NPs have a high curvature and surface-to-mass ratio and corresponding low coordination atoms at the surface, which could enhance dissolution. However, there are many other factors to take into account, such as the metal solubility within a given environment, NP stability and aggregation states, functionalization of NPs with protective shells or coatings, and other properties of the exposure media such as pH, ionic strength, and/or the presence of adsorbing species. Thus NPs may be subjected to a process of disintegration due to chemical reactions with their surroundings—merely from exposure to oxygen in the atmosphere or to certain substances such as chlorine or even enzymes, or simply because the process itself is thermodynamically/kinetically favorable. For the biological context, it is important to note that corrosion is the general term for the natural process by which a material is converted into a more chemically stable form, while biodegradation is recommended by the IUPAC to be limited to the degradation caused by an enzymatic process resulting from the action of cells [77]. In general, organic nanomaterials are more prone to be subjected to biodegradation [78]. The biodegradation of CNTs through enzymatic catalysis has also been described [79]. In contrast, inorganic nanomaterials are more commonly corroded in biological environments by chemical reactions with oxidants such as oxygen or sulfur, or by some other kind of hydrolytic process. For instance, it is known that cysteine dissolves gold [80] and chlorine dissolves gold and silver [81], among others. However, reports hypothesizing disintegration of metal NPs due to enzymatic activity

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in lysozomes have appeared recently. In 2015 Jiang et al. [82] compared the dissolution of AgNPs in acidic media and after endocytosis by epithelial cells. In model media, 7.5% of total Ag was dissolved into ions while 80% was dissolved after endocytosis. Similar effects have been observed in the case of Au, Zn, and FeOx NPs [83]. One important consequence of NP degradation or corrosion, given the importance of size in the nanometric regime, is that corroded particles are not in the same size range anymore when compared to the original materials, the size distribution of the NPs broadens, and it may also affect their morphology (Fig. 1 4). Another important consequence is the toxicity of the associated metal cations. Nowadays, there is an increase in reports establishing relationships between observed effects after NP exposure and NP disintegration [69a,84]. Probably, the most paradigmatic example is the case of nanosilver where the bactericidal effect of AgNPs was found to be correlated to the amount of Ag1 released ions [85]. Another famous case is QDs. Kirchner et al. [84c] and Derfus et al. [69a] showed in the early 2000s how release of Cd ions caused the intracellular oxidation and toxicity of CdSe QDs. Cd binds to sulfhydryl groups of key mitochondrial proteins, leading to cell death. Physiological levels of metallothionein, a protein found in the cytoplasm of hepatocytes which detoxifies Cd by sequestering it into an inert complex, were not sufficient for cells exposed to high levels of Cd21 ions released from QDs. Finally, it is worth mentioning the feromuxytol case [86], an FDA-approved iron oxide NP suspension for anemia treatment, which shows that, in the same way as the NP corrosion phenomenon could result in biological or environmental hazards in some cases, this process could also be harnessed for different applications, such as the delivery of specifically desired compounds (in this case iron ions) for therapy.

FIGURE 1–4 Morphological modifications of NPs over time. TEM images of 8 nm Fe3O4NPs and 15 nm AgNPs as synthesized and after 100 days in cell culture medium. NP, Nanoparticles.

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1.3 Physicochemical Modifications of Inorganic Nanoparticles Determine Their Biodistribution and Fate Once NPs have been exposed to organisms, the response of bodies is diverse. As described in the previous section, the pharmacokinetics—and biological effects—of NPs depend on different parameters: size, shape, surface chemistry and surface properties (area, porosity, charge, coatings), agglomeration state, biopersistence, and dose. These parameters are likely to modify NP fate, such as translocation across epithelia to other organs, binding to proteins and receptors, possible localization in different cellular organelles, induction of oxidative stress, etc. It is worth noting here that, in general, most of the research into NPs in vivo has been carried out in mammalian systems. For nanomedicine, oral and gastrointestinal (GI) tract and i.v. injection have been considered as the most common administration routes; but others such as dermal, nasal, or subcutaneous injection have been also addressed. In the case of nanosafety, inhalation (respiratory system) has been also widely studied as a more likely route of unintentional exposure to nanomaterials. Also, as a general consideration, on exposure to the body, particles of different surface characteristics, size, and morphology attract different arrays of serum proteins and opsonins forming the so-called NP PC. Considerations about NP PC have been extensively reviewed [64,87], and also addressed in this chapter. In vivo, it is reported that complement proteins are likely to bind the NP PC in a process called opsonization, making the particles more susceptible to their removal by the action of phagocytes of the immune system [88]. After this, endocytosis/phagocytosis of the particles, generally by the circulating monocytes or the fixed macrophages, leads to their elimination from the circulation toward organs with high phagocytic activity.

1.3.1 Biodistribution: Nanoparticles Entering the Body As stated earlier, the liver is the major receptor site followed by the spleen, kidneys, and other organs of the reticuloendothelial system. These NP collectors are regenerated within days or few weeks, and then NPs are excreted and disappear from the body. One of the first studies employing nanomaterials that showed these results was made by Nemmar et al. [89]. They used an aerosol consisting mainly of carbon particles of 100 nm radiolabeled with 99mTechnetium (Technegass). They observed that the particles passed through the lung barrier in less than a minute, and reached the liver in less than 1 hour, being accumulated there prior to their final elimination. In another work the same year, Brown et al. [90] reported similar behavior. After these studies and many others, it seems clear that after translocation from the respiratory system, the GI tract, the skin, or i.v. injection, NPs are cleared up rapidly (within minutes) from the bloodstream, with the liver and spleen (  90%) and the kidneys (  9%) being the typical final biodistribution [53]. In general, the larger ones are retained first in the spleen and liver and the smaller ones that pass this filter end up in the kidneys. From there, NPs are expulsed with the feces or urine. As a guide, it is usually considered that continuous

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capillaries found in most tissues, such as muscle, lung, and skin, have a cutoff about 6 nm and fenestrated capillaries in kidney, intestine, and some endocrine and exocrine glands have a cutoff about 50 60 nm. Once the particles are excreted from the body, their final fate, as explained in the previous section, is either dissolution, agglomeration, or absorption onto sediments to be finally immobilized [91]. To overcome these limitations, nanomaterials can be designed so as to circumvent the first-pass effect in the liver. Surface modifications, such as coating with polyethylene glycol (PEG), cell-penetrating peptides, and other targeting molecules, may prevent hepatic and spleen accumulation, opening the possibility of reaching other organs. The possibilities that NP functionalization opens for targeted nanomedicine have been discussed above. In addition, subcutaneous or intratumoral injection is proposed as the most promising route for successful implementation in patients, and they are currently the most frequently used route in animal experimentation [92]. This route of administration overcomes the limitations arising from systemic circulation, and in the case of oncology, targeting can also be optimized by delivering the nanomaterial directly to the interstitium of the cancerous tissue [93], since the interstitial pressure in tumors seems to be higher than in healthy tissues, enabling greater leakage of the drugs [94]. Again, the fate of the nanomaterials after subcutaneous injection heavily depends on physicochemical properties of the nanomaterials in the conditions of the interstitial lymphatic flow. Note that despite its advantages and successes, intratumoral injection is only applicable for easily accessible tumors [92].

1.3.2 Subcellular Localization: Nanoparticles Entering the Cells Once NPs have entered the body, and have been distributed, the next step is to investigate their penetration into cells after crossing the cytoplasmic membrane. This membrane controls entry into the cell and has a crucial role in development, uptake of nutrients, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organism homeostasis. For this, the different uptake mechanisms of different substances have been widely studied. These mechanisms mostly depend on the size of the object to be internalized. Small molecules, such as ions, amino acids, or sugars cross the cell through pinocytosis, via membrane protein pumps or channels, while larger molecular entities enter the cell via endocytosis, in membrane-bound vesicles formed by invagination of the plasma membrane. Endocytosis can be active (receptor-mediated) or passive (by adhesive interaction and invagination of the membrane). Whichever the endocytic routes of uptake (receptor-mediated, macropinocytosis, micropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin- and caveolae-independent endocytosis; see, for example, Ref. [95]), the material initially remains in a subcellular compartment, the endosome, which is still separated from the cytoplasm of the cell by a membrane. It is very difficult to draw a map of the mechanism of NP internalization into cells since the endocytosis process for nanomaterials and other foreign materials is very diverse. The uptake mechanism activated will depend on the characteristics of the material (size, shape, composition, surface coating) the cell type (e.g., Refs. [96,87], showing different uptake

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FIGURE 1–5 CeO2 NPs internalized by human hepatocytes (HepG2 cells). TEM images of hepatocytes revealing internalization of the NPs and localization in the cytoplasm. (Left) Bright field images and (right) dark field images of the same area allowing NPs to be easily distinguished. NP, Nanoparticles.

mechanisms for different nanomaterials) and the physical interaction of the material with the cellular membrane [97]. In addition, the chemical and physical properties of the cellular membrane responsible for the translocation of nanomaterials into cells, the nucleus, and organelles are still unknown [92]. As a general consideration, it is important to note that the vast majority of studies showed NP presence inside vesicular structures and not the cytosol (Fig. 1 5). Release from endosomes and reaching the cytosol is challenging. Some of the strategies performed to obtain cytoplasmatic release from the endosomes are the use of disrupting peptides such as the mentioned Tat-peptide [98], or the proton sponge mechanism [99]. What is known is that most of these endocytic routes end up in the lysosome and/or exosomes. Lysosomes are the degradative compartment of the cell where materials are exposed to high concentrations of a wide variety of hydrolytic enzymes and acidic pH, as discussed in Section 1.2. In this sense, NPs do not remain for a long time inside those vesicles, either NPs are dissolved or they are expulsed again in an exosome. In the case that NPs remain inside the cell, their fate will be bound to that of the cell. And since cellular recycling in the body is continuously regenerating, NP permanence in the body will be limited and disperse in time. The cell turnover rate varies from weeks in tissues such as the skin and GI tract, to years in bone or neurons. In any case, so far, reported cases of extended permanence of inorganic matter in the body are granulomatosis (such as silicosis, asbestosis, etc.), where macrophages are not able to internalize and degrade micrometric particles which ultimately leads to chronic inflammation (and cancer) as was shown for the mentioned case of .10 nm CNTs [56]. Thus these events are not likely to apply in the case of well-stabilized inorganic NPs, since they are much smaller and can be easily internalized by macrophages.

1.3.3 Long-Term Effects There is still not an extensive literature on the fate of NPs intended for medical applications over more than a few days and data are scarce. This could be most likely due to the limited

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characterization possibilities for tracking nanomaterials over long periods of time and the maintenance of animal models [92]. It is important since in the case some NPs stay for long periods of time inside cells or organisms, even at nontoxic concentrations, they may have side effects. Thus long-term chronic and repetitive exposure should be considered [53]. It should be carefully investigated how cells respond to treatments with nanomaterials below the doses causing a high percentage of cell death since even small changes may cause profound effects on the integrity and viability of the cells over multiple cellular divisions. Probably due to those difficulties, data on the long-term consequences of exposure to inorganic NPs in the body are not only scarce but sometimes also contradictory. In a recent study carried out by Wang et al. [100] using 3.5 nm AuNPs, the presence of Au in kidneys and accumulation in tumor tissue in mice were reported for a period of 90 days. In contrast Naz et al. [101], using 2, 5, and 10 nm AuNPs also for a period of 90 days, found complete elimination of all sizes. Of course, for larger sizes, data are more concise and indicate that, although with some rates of clearance, there is still a detectable presence of NPs or NP debris at least after a number of months. For instance, Sadauskas et al. [102] using 40 nm AuNPs found Au in the liver of mice after 6 months. Similarly, Goel et al. [103], using 27 nm PEG-coated AuNPs found Au in the tumor interstitium in mice after 120 days as well as in the spleen, liver, and kidneys. Other inorganic NPs of interest in medicine may be more prone to dissolution and clearance through the kidneys after a few weeks, such as some small (,10 nm) metal oxides [32d,104], but still some reports found persistence of NPs in the case of large ( . 30 nm) FeOx [105] and SiO2 [106], among others. Both these results and the lack of enough data on long-term exposure call for the need for studies into the chronic implications of the use of NPs in medicine.

1.4 Outlook and Conclusion NPs are unique tools for the successful application of novel nanotechnologies in health. These applications have been growing progressively over the last two decades and are still raising high expectations for better, more efficient, and affordable health care. On the one hand, in this chapter, we have pointed out how the first nanotechnology-based medical solutions are already on the market, many are in clinical trials, but most of the future promising applications are still under development. The specificity of functionalized NPs for targeting at tissue and cellular levels, in both diagnostic imaging and drug-based therapies, is pushing research forward to create novel theranostic nanoplatforms that will, in turn, prevent nonspecific cell binding in healthy tissues (personalized medicine with less/no side effects). For instance, in diagnosis, hand-held devices with highly accurate, highly sensitive, multiplexed, and inexpensive testing are already in the pipeline for many biotech companies. In addition, molecularly targeted NPs with different labels offer many advantages over conventional molecular imaging probes. Also, a combination of labels for different imaging modalities can be attached to a single NP, and at the same time, the same NP can contain different targeting ligands which provide enhanced receptor binding affinity or specificity. In therapy, for

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instance, targeted medical applications are the focus of the European Technology Platform of Nanomedicine where nanomedicine will have a significant impact, including in Alzheimer’s disease, cancer, ophthalmology, combating antimicrobial resistance, diabetes, infectious diseases, atherosclerosis, arthritis, and tissue engineering. On the other hand, in this chapter we have reviewed different studies of biodistribution and the fate of inorganic NPs for nanomedicine and nanosafety that show how the differently observed biological fates and effects are mainly related to its evolution (agglomeration, dissolution, protein adsorption) in the physiological media, making this a crucial area of research that will allow for an efficient implementation of nanomedicine. It is accepted that NPs may be destabilized when traveling through different parts of the body. Their high surface energy tends to aggregate them homogeneously (forming polycrystalline particles) or heterogeneously (with molecules and structures of the surroundings) [63], both altering and modifying the biodistribution. Similarly, during their time inside the body, NPs are affected by the presence of different redox states (from rather reducing to clearly oxidizing), pH (the late endosomes and lysozomes can go down to 5), and the presence of nucleophilic species and ionic scavengers. Inside the body, protein absorption onto the NP surface not only modifies NP surface properties but also may result in protein changes and alter their metabolization as preliminary results have shown [107]. The consequences of these modifications in protein conformation and metabolization in, for example, the immune response are still generally unknown. Importantly, all these modifications depend to a large extent on the characteristics of the biological media in which the NPs are dispersed, and they are underappreciated parameters that need to be carefully addressed to better understand NP effects. The development of reproducible and reliable analytical methods for the intracellular, tissue-, and organ-specific characterization of nanomaterial evolution is still a challenge, and limited data are available. However, ultimately, the knowledge of these pharmacokinetic and biodistribution aspects that drive NP behavior and effects will allow further advantage to be taken of NPs’ potential clinical benefits.

References [1] E. Casals, M.F. Gusta, M. Cobaleda-Siles, A. Garcia-Sanz, V.F. Puntes, Cancer resistance to treatment and antiresistance tools offered by multimodal multifunctional nanoparticles, Cancer Nanotechnol. 8 (1) (2017) 7. [2] J. Xie, S. Lee, X. Chen, Nanoparticle-based theranostic agents, Adv. Drug Deliv. Rev. 62 (11) (2010) 1064 1079. [3] (a) A.P. Alivisatos, Less is more in medicine, Sci. Am. 285 (3) (2001) 66 73. (b) R.F. Service, Materials and biology. Nanotechnology takes aim at cancer, Science 310 (5751) (2005) 1132 1134. [4] Y. Mi, Y. Guo, S.S. Feng, Nanomedicine for multimodality treatment of cancer, Nanomedicine (Lond.) 7 (12) (2012) 1791 1794. [5] R.A. Sperling, E. Casals, J. Comenge, N.G. Bastus, V.F. Puntes, Inorganic engineered nanoparticles and their impact on the immune response, Curr. Drug Metabol. 10 (8) (2009) 895 904.

20

THERANOSTIC BIONANOMATERIALS

[6] (a) N.G. Bastus, J. Comenge, V. Puntes, Kinetically controlled seeded growth synthesis of citratestabilized gold nanoparticles of up to 200 nm: size focusing versus Ostwald ripening, Langmuir 27 (17) (2011) 11098 11105. (b) E. Gonzalez, J. Arbiol, V.F. Puntes, Carving at the nanoscale: sequential galvanic exchange and Kirkendall growth at room temperature, Science 334 (6061) (2011) 1377 1380. [7] X.J. Liang, C. Chen, Y. Zhao, P.C. Wang, Circumventing tumor resistance to chemotherapy by nanotechnology, Methods Mol. Biol. 596 (2010) 467 488. [8] K.S. Moulton, B.R. Olsen, S. Sonn, N. Fukai, D. Zurakowski, X. Zeng, Loss of collagen XVIII enhances neovascularization and vascular permeability in atherosclerosis, Circulation 110 (10) (2004) 1330 1336. [9] S. Barua, S. Mitragotri, Challenges associated with penetration of nanoparticles across cell and tissue barriers: a review of current status and future prospects, Nano Today 9 (2) (2014) 223 243. [10] S. Bhattacharyya, R. Bhattacharya, S. Curley, M.A. McNiven, P. Mukherjee, Nanoconjugation modulates the trafficking and mechanism of antibody induced receptor endocytosis, Proc. Natl. Acad. Sci. U.S.A. 107 (33) (2010) 14541 14546. [11] J. Comenge, C. Sotelo, F. Romero, O. Gallego, A. Barnadas, T.G. Parada, et al., Detoxifying antitumoral drugs via nanoconjugation: the case of gold nanoparticles and cisplatin, PLoS One 7 (10) (2012) e47562. [12] (a) V. Puntes, Design and pharmacokinetical aspects for the use of inorganic nanoparticles in radiomedicine, Br. J. Radiol. 89 (1057) (2016) 20150210.(b) V. Puntes, J. Saldaña, Nano-Oncology, the Turning Point: Discover the Wave of Knowledge That Makes Fighting Cancer With Nanotechnology Real. nanowiki edicions 2015. [13] (a) A. Espinosa, A.K. Silva, A. Sanchez-Iglesias, M. Grzelczak, C. Pechoux, K. Desboeufs, et al., Cancer cell internalization of gold nanostars impacts their photothermal efficiency in vitro and in vivo: toward a plasmonic thermal fingerprint in tumoral environment, Adv. Healthcare Mater. 5 (9) (2016) 1040 1048. (b) Y. Liu, H. Yuan, A.M. Fales, J.K. Register, T. Vo-Dinh, Multifunctional gold nanostars for molecular imaging and cancer therapy, Front. Chem. 3 (2015) 51. (c) J. Nam, S. Son, L.J. Ochyl, R. Kuai, A. Schwendeman, J.J. Moon, Chemo-photothermal therapy combination elicits anti-tumor immunity against advanced metastatic cancer, Nat. Commun. 9 (1) (2018) 1074. [14] B.R. Smith, P. Kempen, D. Bouley, A. Xu, Z. Liu, N. Melosh, et al., Shape matters: intravital microscopy reveals surprising geometrical dependence for nanoparticles in tumor models of extravasation, Nano Lett. 12 (7) (2012) 3369 3377. [15] (a) D.S. Karaman, D. Desai, R. Senthilkumar, E.M. Johansson, N. Råtts, M. Odén, et al., Shape engineering vs organic modification of inorganic nanoparticles as a tool for enhancing cellular internalization, Nanoscale Res. Lett. 7 (1) (2012) 358. (b) D.S. Karaman, S. Sarwar, D. Desai, E.M. Björk, M. Odén, P. Chakrabarti, et al., Shape engineering boosts antibacterial activity of chitosan coated mesoporous silica nanoparticle doped with silver: a mechanistic investigation, J. Mater. Chem. B 4 (19) (2016) 3292 3304. [16] A.D. Frankel, C.O. Pabo, Cellular uptake of the tat protein from human immunodeficiency virus, Cell 55 (6) (1988) 1189 1193. [17] J.B. Delehanty, K. Boeneman, C.E. Bradburne, K. Robertson, J.E. Bongard, I.L. Medintz, Peptides for specific intracellular delivery and targeting of nanoparticles: implications for developing nanoparticlemediated drug delivery, Ther. Deliv. 1 (3) (2010) 411 433. [18] (a) N.G. Bastus, E. Sanchez-Tillo, S. Pujals, C. Farrera, M.J. Kogan, E. Giralt, et al., Peptides conjugated to gold nanoparticles induce macrophage activation, Mol. Immunol. 46 (4) (2009) 743 748. (b) N.G. Bastus, E. Sanchez-Tillo, S. Pujals, C. Farrera, C. Lopez, E. Giralt, et al., Homogeneous conjugation of peptides onto gold nanoparticles enhances macrophage response, ACS Nano 3 (6) (2009). 1335-44. (c) Z.R. Cui, S.J. Han, D.P. Vangasseri, L. Huang, Immunostimulation mechanism of LPD nanoparticle as a vaccine carrier, Mol. Pharm. 2 (1) (2005) 22 28.

Chapter 1 • Biodistribution, Excretion, and Toxicity of Inorganic Nanoparticles

21

[19] L. Garcia-Fernandez, J. Garcia-Pardo, O. Tort, I. Prior, M. Brust, E. Casals, et al., Conserved effects and altered trafficking of Cetuximab antibodies conjugated to gold nanoparticles with precise control of their number and orientation, Nanoscale 9 (18) (2017) 6111 6121. [20] J.A. Hubbell, S.N. Thomas, M.A. Swartz, Materials engineering for immunomodulation, Nature 462 (7272) (2009) 449 460. [21] C. Chavany, T. Saisonbehmoaras, T. Ledoan, F. Puisieux, P. Couvreur, C. Helene, Adsorption of oligonucleotides onto polyisohexylcyanoacrylate nanoparticles protects them against nucleases and increases their cellular uptake, Pharm. Res. 11 (9) (1994) 1370 1378. [22] E. Casals, T. Pfaller, A. Duschl, G.J. Oostingh, V. Puntes, Hardening of the nanoparticle protein corona in metal (Au, Ag) and oxide (Fe3O4, CoO and CeO2) nanoparticles, Small (2011). Available from: https:// doi.org/10.1002/smll.201101511 (Article first published online: November 7, 2011). [23] J.A. O’Shaughnessy, S. Tjulandin, N. Davidson, ABI-007 (ABRAXANE), a nanoparticle albumin-bound (nab) paclitaxel demonstrates superior efficacy vs taxol in MBC: a phase III trial, in: Paper Presented at the 26th Annual San Antonio Breast Cancer Symposium, December 3 6, 2003, Abstract 44. [24] P.K. Jain, M.A. El-Sayed, Surface plasmon resonance sensitivity of metal nanostructures: physical basis and universal scaling in metal nanoshells, J. Phys. Chem. C 111 (47) (2007) 17451 17454. [25] B. Nikoobakht, M.A. El-Sayed, Preparation and growth mechanism of gold nanorods (NRs) using seedmediated growth method, Chem. Mater. 15 (10) (2003) 1957 1962. [26] P. Senthil Kumar, I. Pastoriza-Santos, B. Rodriguez-Gonzalez, F. Javier Garcia de Abajo, L.M. LizMarzan, High-yield synthesis and optical response of gold nanostars, Nanotechnology 19 (1) (2008) 015606. [27] S.E. Skrabalak, J. Chen, Y. Sun, X. Lu, L. Au, C.M. Cobley, et al., Gold nanocages: synthesis, properties, and applications, Acc. Chem. Res. 41 (12) (2008) 1587 1595. [28] P.K. Jain, I.H. El-Sayed, M.A. El-Sayed, Au nanoparticles target cancer, Nano Today 2 (1) (2007) 18 29. [29] (a) F.E. Auzel, Materials and devices using double-pumped phosphors with energy transfer, Proc. IEEE 61 (6) (1973) 758 785. (b) F.W. Ostermayer, Preparation and properties of infrared-to-visible conversion phosphors, Metall. Trans. 2 (3) (1971) 747 755. [30] (a) M. Haase, H. Schafer, Upconverting nanoparticles, Angew. Chem. 50 (26) (2011) 5808 5829. (b) K.L. Reddy, N. Prabhakar, R. Arppe, J.M. Rosenholm, V. Krishnan, Microwave-assisted one-step synthesis of acetate-capped NaYF4:Yb/Er upconversion nanocrystals and their application in bioimaging, J. Mater. Sci. 52 (10) (2017) 5738 5750. (c) K.L. Reddy, et al., Controlled synthesis, bioimaging and toxicity assessments in strong red emitting Mn2 1 doped NaYF4:Yb3 1 /Ho3 1 nanophosphors, RSC Adv. 6 (59) (2016) 53698 53704. [31] (a) A.J. Giustini, A.A. Petryk, S.M. Cassim, J.A. Tate, I. Baker, P.J. Hoopes, Magnetic nanoparticle hyperthermia in cancer treatment, Nano LIFE 1 (1n02) (2010). (b) A.K. Gupta, et al., PEI-PEG-chitosan copolymer coated iron oxide nanoparticles for safe gene delivery: synthesis, complexation, and transfection, Adv. Funct. Mater. 19 (14) (2009) 2244 2251. [32] (a) J.P. Chen, S. Patil, S. Seal, J.F. McGinnis, Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides, Nat. Nanotechnol. 1 (2) (2006) 142 150. (b) S. Hirst, A. Karakoti, R. Tyler, et al., Cerium oxide nanoparticles reduce steatosis, portal hypertension and display anti-inflammatory properties in rats with liver fibrosis, J. Hepatol. 64 (3) (2016) 691 698. [33] (a) Y. Barenholz, Doxils—the first FDA-approved nano-drug: lessons learned, J. Controlled Release 160 (2) (2012) 117 134.

22

THERANOSTIC BIONANOMATERIALS

(b) A. Gabizon, D. Goren, Z. Fuks, Y. Barenholz, A. Dagan, A. Meshorer, Enhancement of adriamycin delivery to liver metastatic cells with increased tumoricidal effect using liposomes as drug carriers, Cancer Res. 43 (10) (1983) 4730 4735. (c) A. Gabizon, et al., Superior therapeutic activity of liposome-associated adriamycin in a murine metastatic tumour model, Br. J. Cancer 51 (5) (1985) 681 689. [34] N. Prabhakar, T. Näreoja, E. von Haartman, D.¸S. Karaman, H. Jiang, S. Koho, et al., Core shell designs of photoluminescent nanodiamonds with porous silica coatings for bioimaging and drug delivery II: application, Nanoscale 5 (9) (2013) 3713 3722. [35] (a) G. Chen, H. Qiu, P.N. Prasad, X. Chen, Upconversion nanoparticles: design, nanochemistry, and applications in theranostics, Chem. Rev. 114 (10) (2014) 5161 5214. (b) C.T. Xu, Q. Zhan, H. Liu, et al., Upconverting nanoparticles for pre-clinical diffuse optical imaging, microscopy and sensing: current trends and future challenges, Laser Photonics Rev. 7 (5) (2013) 663 697. [36] A.J. Raghavendra, W.E. Gregory, T.J. Slonecki, Y. Dong, I. Persaud, J.M. Brown, et al., Three-photon imaging using defect-induced photoluminescence in biocompatible ZnO nanoparticles, Int. J. Nanomed. 13 (2018) 4283 4290. [37] Y.-X. Zhang, Y.-H. Wang, Nonlinear optical properties of metal nanoparticles: a review, RSC Adv. 7 (71) (2017) 45129 45144. [38] (a) N. Nicholas, D.A. Marta, P. Neeraj, M.P. Alexander, F. Natalya, D.T. Marco, et al., Fluorescent single-digit detonation nanodiamond for biomedical applications, Methods Appl. Fluoresc. 6 (3) (2018) 035010. (b) N. Prabhakar, M. Peurla, S. Koho, T. Deguchi, T. Näreoja, H.-C. Chang, et al., STED-TEM correlative microscopy leveraging nanodiamonds as intracellular dual-contrast markers, Small 14 (5) (2018) 1701807. [39] (a) D.¸S. Karaman, M.P. Sarparanta, J.M. Rosenholm, A.J. Airaksinen, Multimodality imaging of silica and silicon materials in vivo, Adv. Mater. 30 (24) (2018) 1703651. (b) J.M. Rosenholm, T. Gulin-Sarfraz, V. Mamaeva, et al., Prolonged dye release from mesoporous silica-based imaging probes facilitates long-term optical tracking of cell populations in vivo, Small 12 (12) (2016) 1578 1592. [40] (a) R. Juliano, Nanomedicine- is the wave cresting? Nat. Rev. Drug Discov. 12 (3) (2013) 171 172. (b) V.J. Venditto, F.C. Szoka, Cancer nanomedicines: so many papers and so few drugs!, Adv. Drug Deliv. Rev. 65 (1) (2013) 80 88. [41] D. Bobo, K.J. Robinson, J. Islam, K.J. Thurecht, S.R. Corrie, Nanoparticle-based medicines: a review of FDA-approved materials and clinical trials to date, Pharm. Res. 33 (10) (2016) 2373 2387. [42] E. Casals, M.F. Gusta, J. Piella, G. Casals, W. Jiménez, V. Puntes, Intrinsic and extrinsic properties affecting innate immune responses to nanoparticles: the case of cerium oxide, Front. Immunol. 8 (2017) 970. [43] (a) Join the dialogue, Nat. Nanotechnol. 7 (2012) 545. (b) H.F. Krug, Nanosafety research—are we on the right track? Angew. Chem. Int. Ed. 53 (46) (2014) 12304 12319. [44] (a) M. Fisichella, F. Berenguer, G. Steinmetz, M. Auffan, J. Rose, O. Prat, Toxicity evaluation of manufactured CeO(2) nanoparticles before and after alteration: combined physicochemical and whole-genome expression analysis in Caco-2 cells, BMC Genomics 15 (1) (2014) 700. (b) S.K. Nalabotu, et al., Intratracheal instillation of cerium oxide nanoparticles induces hepatic toxicity in male Sprague-Dawley rats, Int. J. Nanomed. 6 (2011) 2327 2335. [45] (a) D. Oro, G. Fernandez-Varo, V. Reichenbach, T. Yudina, E. Casals, G. Casals, et al., Cerium oxide nanoparticles reduce portal hypertension and show antiinflammatory properties in CCl4-treated rats, Hepatology 60 (2014) 1175A.

Chapter 1 • Biodistribution, Excretion, and Toxicity of Inorganic Nanoparticles

23

(b) G.F. Varo, D. Oró, S.C. Restoy, L. Boix, E. Casals, M.P. Corominas, et al., CeO2NPs are similarly effective as sorafenib in increasing survival in rats with HCC, J. Hepatol. 68 (2018) S662 S663. (c) R.A. Yokel, M.T. Tseng, M. Dan, J.M. Unrine, U.M. Graham, P. Wu, et al., Biodistribution and biopersistence of ceria engineered nanomaterials: size dependence, Nanomed.: Nanotechnol. Biol. Med. 9 (3) (2013) 398 407. [46] (a) S. Aalapati, S. Ganapathy, S. Manapuram, G. Anumolu, B.M. Prakya, Toxicity and bio-accumulation of inhaled cerium oxide nanoparticles in CD1 mice, Nanotoxicology 8 (7) (2014) 786 798. (b) A. Srinivas, P.J. Rao, et al., Distribution, elimination, and biopersistence to 90 days of a systemically introduced 30 nm ceria-engineered nanomaterial in rats, Toxicol. Sci. 127 (1) (2012) 256 268. [47] (a) R. Weissleder, G. Elizondo, J. Wittenberg, C.A. Rabito, H.H. Bengele, L. Josephson, Ultrasmall superparamagnetic iron oxide: characterization of a new class of contrast agents for MR imaging, Radiology 175 (2) (1990) 489 493. (b) R. Weissleder, et al., Superparamagnetic iron oxide: pharmacokinetics and toxicity, Am. J. Roentgenol. 152 (1) (1989) 167 173. [48] E.H. Sciences, Nanomedicine opens the way for nerve cell regeneration. ,https://www.sciencedaily. com/releases/2007/05/070520091842.htm.. [49] T.R. Pisanic, J.D. Blackwell, V.I. Shubayev, R.R. Fiñones, S. Jin, Nanotoxicity of iron oxide nanoparticle internalization in growing neurons, Biomaterials 28 (16) (2007) 2572 2581. [50] E. Casals, E. Gonzalez, V.F. Puntes, Reactivity of inorganic nanoparticles in biological environments: insights into nanotoxicity mechanisms, J. Phys. D: Appl. Phys. 45 (44) (2012) 443001. [51] E. Cho, Q. Zhang, Y. Xia, The effect of sedimentation and diffusion on cellular uptake of gold nanoparticles, Nat. Nanotechnol. 6 (6) (2011) 385 391. [52] E. Casals, T. Pfaller, A. Duschl, G.J. Oostingh, V. Puntes, Time evolution of the nanoparticle protein corona, ACS Nano 4 (7) (2010) 3623 3632. [53] E. Casals, S. Vázquez-Campos, N.G. Bastús, V. Puntes, Distribution and potential toxicity of engineered inorganic nanoparticles and carbon nanostructures in biological systems, TrAC Trends Anal. Chem. 27 (8) (2008) 672 683. [54] G. Oberdörster, E. Oberdörster, J. Oberdörster, Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles, Environ. Health Perspect. 113 (7) (2005) 823 839. [55] L. Braydich-Stolle, S. Hussain, J.J. Schlager, M.-C. Hofmann, In vitro cytotoxicity of nanoparticles in mammalian germline stem cells, Toxicol. Sci. 88 (2) (2005) 412 419. [56] C.A. Poland, R. Duffin, I. Kinloch, A. Maynard, W.A.H. Wallace, A. Seaton, et al., Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study, Nat. Nanotechnol. 3 (2008) 423. [57] E.H. Anne, R. Heiko, Spatial organization of the cytoskeleton enhances cargo delivery to specific target areas on the plasma membrane of spherical cells, Phys. Biol. 13 (6) (2016) 066003. [58] (a) T.A. Horbett, J.L. Brash, Proteins at Interfaces II, vol. 602, American Chemical Society, 1995, p. 580. (b) W. Norde, J. Lyklema, Why proteins prefer interfaces, J. Biomater. Sci. 2 (3) (1991) 183 202. [59] (a) E.F. Leonard, L. Vroman, Is the Vroman effect of importance in the interaction of blood with artificial materials? J. Biomater. Sci. 3 (1) (1992) 95 107. (b) L. Vroman, A. Adams, G. Fischer, P. Munoz, Interaction of high molecular weight kininogen, factor XII, and fibrinogen in plasma at interfaces, Blood 55 (1) (1980) 156 159. [60] L. Vroman, Effect of adsorbed proteins on wettability of hydrophilic and hydrophobic solids, Nature 196 (4853) (1962). 476 &. [61] S.M. Slack, T.A. Horbett, The Vroman effect—a critical review, Proteins Interfaces II 602 (1995) 112 128.

24

THERANOSTIC BIONANOMATERIALS

[62] S. Alaeddine, H. Nygren, Logarithmic growth of protein films, in: T.A. Horbett, J.L. Brash (Eds.), Proteins at Interfaces II. Fundamentals and Applications, Vol. 602, ACS, Washington, DC, 1995. [63] F. Barbero, L. Russo, M. Vitali, J. Piella, I. Salvo, M.L. Borrajo, et al., Formation of the protein corona: the interface between nanoparticles and the immune system, Semin. Immunol. 34 (2017) 52 60. [64] (a) E. Casals, V.F. Puntes, Inorganic nanoparticle biomolecular corona: formation, evolution and biological impact, Nanomedicine 7 (12) (2012) 1917 1930. (b) M. Mahmoudi, I. Lynch, M.R. Ejtehadi, M.P. Monopoli, F.B. Bombelli, et al., Protein 2 nanoparticle interactions: opportunities and challenges, Chem. Rev. 111 (9) (2011) 5610 5637. [65] D. Walczyk, F.B. Bombelli, M.P. Monopoli, I. Lynch, K.A. Dawson, What the cell “sees” in bionanoscience, J. Am. Chem. Soc. 132 (16) (2010) 5761 5768. [66] (a) M.A. Dobrovolskaia, A.K. Patri, J.W. Zheng, J.D. Clogston, N. Ayub, P. Aggarwal, et al., Interaction of colloidal gold nanoparticles with human blood: effects on particle size and analysis of plasma protein binding profiles, Nanomed. Nanotechnol. Biol. Med. 5 (2) (2009) 106 117. (b) S.H.D. Lacerda, J.J. Park, C. Meuse, D. Pristinski, M.L. Becker, A. Karim, et al., Interaction of gold nanoparticles with common human blood proteins, ACS Nano 4 (1) (2010) 365 379. [67] C. Rocker, M. Potzl, F. Zhang, W.J. Parak, G.U. Nienhaus, A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles, Nat. Nanotechnol. 4 (9) (2009) 577 580. [68] Z.J. Deng, G. Mortimer, T. Schiller, A. Musumeci, D. Martin, R.F. Minchin, Differential plasma protein binding to metal oxide nanoparticles, Nanotechnology 20 (2009) 45. [69] (a) A.M. Derfus, W.C.W. Chan, S.N. Bhatia, Probing the cytotoxicity of semiconductor quantum dots, Nano Lett. 4 (1) (2004) 11 18. (b) B. Sahoo, M. Goswami, S. Nag, S. Maiti, Spontaneous formation of a protein corona prevents the loss of quantum dot fluorescence in physiological buffers, Chem. Phys. Lett. 445 (4-6) (2007) 217 220. [70] P. Ebbesen, DEAE-dextran and polybrene cation enhancement and dextran sulfate anion inhibition of immune cytolysis, J. Immunol. 109 (6) (1972) 1296 1299. [71] P.H.M. Hoet, L. Gilissen, B. Nemery, Polyanions protect against the in vitro pulmonary toxicity of polycationic paint components associated with the ardystil syndrome, Toxicol. Appl. Pharmacol. 175 (2) (2001) 184 190. [72] A. Hoshino, K. Fujioka, T. Oku, M. Suga, Y.F. Sasaki, T. Ohta, et al., Physicochemical properties and cellular toxicity of nanocrystal quantum dots depend on their surface modification, Nano Lett. 4 (11) (2004) 2163 2169. [73] C.M. Goodman, C.D. McCusker, T. Yilmaz, V.M. Rotello, Toxicity of gold nanoparticles functionalized with cationic and anionic side chains, Bioconjug. Chem. 15 (4) (2004) 897 900. [74] L. Balogh, S.S. Nigavekar, B.M. Nair, W. Lesniak, C. Zhang, L.Y. Sung, et al., Significant effect of size on the in vivo biodistribution of gold composite nanodevices in mouse tumor models, Nanomedicine 3 (4) (2007) 281 296. [75] A. Ito, X. Sun, T. Tateishi, In-vitro analysis of metallic particles, colloidal nanoparticles and ions in wear-corrosion products of SUS317L stainless steel, Mater. Sci. Eng. C 17 (2001) 161 166. [76] (a) A.E. Goode, J.M. Perkins, A. Sandison, C. Karunakaran, H. Cheng, D. Wall, et al., Chemical speciation of nanoparticles surrounding metal-on-metal hips, Chem. Commun. 48 (67) (2012) 8335 8337. (b) J. Kolosnjaj-Tabi, L. Lartigue, Y. Javed, N. Luciani, T. Pellegrino, C. Wilhelm, et al., Biotransformations of magnetic nanoparticles in the body, Nano Today 11 (3) (2016) 280 284. [77] M. Vert, Y. Doi, K.-H. Hellwich, M. Hess, P. Hodge, P. Kubisa, et al., Terminology for biorelated polymers and applications (IUPAC Recommendations 2012), Pure Appl. Chem. Vol. 84 (2012) 377. [78] J.G. Croissant, Y. Fatieiev, N.M. Khashab, Degradability and clearance of silicon, organosilica, silsesquioxane, silica mixed oxide, and mesoporous silica nanoparticles, Adv. Mater. 29 (9) (2017) 1604634.

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[79] B.L. Allen, P.D. Kichambare, P. Gou, I.I. Vlasova, A.A. Kapralov, N. Konduru, et al., Biodegradation of single-walled carbon nanotubes through enzymatic catalysis, Nano Lett. 8 (11) (2008) 3899 3903. [80] A.R. Timerbaev, S.S. Aleksenko, K. Polec-Pawlak, R. Ruzik, O. Semenova, C.G. Hartinger, et al., Platinum metallodrug-protein binding studies by capillary electrophoresis-inductively coupled plasma-mass spectrometry: characterization of interactions between Pt(II) complexes and human serum albumin, Electrophoresis 25 (13) (2004) 1988 1995. [81] K.-H. Kim, J.-U. Kim, S.-H. Cha, J.-C. Lee, Reversible formation and dissolution of gold nanoparticles through turning on and off sequences of UV light, J. Am. Chem. Soc. 131 (22) (2009) 7482 7483. [82] X. Jiang, T. Micl˘au¸s, L. Wang, R. Foldbjerg, D.S. Sutherland, H. Autrup, et al., Fast intracellular dissolution and persistent cellular uptake of silver nanoparticles in CHO-K1 cells: implication for cytotoxicity, Nanotoxicology 9 (2) (2015) 181 189. [83] S. Sabella, R.P. Carney, V. Brunetti, M.A. Malvindi, N. Al-Juffali, G. Vecchio, et al., A general mechanism for intracellular toxicity of metal-containing nanoparticles, Nanoscale 6 (12) (2014) 7052 7061. [84] (a) V. De Matteis, M.A. Malvindi, A. Galeone, V. Brunetti, E. De Luca, S. Kote, et al., Negligible particlespecific toxicity mechanism of silver nanoparticles: the role of Ag 1 ion release in the cytosol, Nanomed.: Nanotechnol. Biol. Med. 11 (3) (2015) 731 739. (b) A. Gnach, T. Lipinski, A. Bednarkiewicz, J. Rybka, J.A. Capobianco, Upconverting nanoparticles: assessing the toxicity, Chem. Soc. Rev. 44 (6) (2015) 1561 1584. (c) C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. Muñoz Javier, H.E. Gaub, et al., Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles, Nano Lett. 5 (2) (2005) 331 338. (d) D. Wang, Z. Lin, T. Wang, Z. Yao, M. Qin, S. Zheng, et al., Where does the toxicity of metal oxide nanoparticles come from: the nanoparticles, the ions, or a combination of both? J. Hazard. Mater. 308 (2016) 328 334. [85] B. Nowack, H.F. Krug, M. Height, 120 Years of nanosilver history: implications for policy makers, Environ. Sci. Technol. 45 (4) (2011) 1177 1183. [86] (a) www.feraheme.com. (b) R. Landry, P.M. Jacobs, R. Davis, M. Shenouda, W.K. Bolton, Pharmacokinetic study of ferumoxytol: a new iron replacement therapy in normal subjects and hemodialysis patients, Am. J. Nephrol. 25 (4) (2005) 400 410. (c) M.H. Schwenk, Ferumoxytol: a new intravenous iron preparation for the treatment of iron deficiency anemia in patients with chronic kidney disease, Pharmacotherapy 30 (1) (2010). 70-9. [87] M. Mahmoudi, S.N. Saeedi-Eslami, M.A. Shokrgozar, K. Azadmanesh, M. Hassanlou, H.R. Kalhor, et al., Cell “vision”: complementary factor of protein corona in nanotoxicology, Nanoscale 4 (17) (2012) 5461 5468. [88] F. Chen, G. Wang, J.I. Griffin, B. Brenneman, N.K. Banda, V.M. Holers, et al., Complement proteins bind to nanoparticle protein corona and undergo dynamic exchange in vivo, Nat. Nanotechnol. 12 (2016) 387. [89] A. Nemmar, et al., Passage of inhaled particles into the blood circulation in humans, Circulation 105 (4) (2002) 411 414. [90] J.S. Brown, K.L. Zeman, W.D. Bennett, Ultrafine particle deposition and clearance in the healthy and obstructed lung, Am. J. Respir. Crit. Care Med. 166 (9) (2002) 1240 1247. [91] M.E. Åkerman, W.C.W. Chan, P. Laakkonen, S.N. Bhatia, E. Ruoslahti, Nanocrystal targeting in vivo, Proc. Natl. Acad. Sci. U.S.A. 99 (20) (2002) 12617 12621. [92] J. Bourquin, A. Milosevic, D. Hauser, R. Lehner, F. Blank, A. Petri-Fink, et al., Biodistribution, clearance, and long-term fate of clinically relevant nanomaterials, Adv. Mater. 30 (19) (2018) 1704307. [93] I. Brigger, C. Dubernet, P. Couvreur, Nanoparticles in cancer therapy and diagnosis, Adv. Drug Deliv. Rev. 54 (5) (2002) 631 651.

26

THERANOSTIC BIONANOMATERIALS

[94] C.-H. Heldin, K. Rubin, K. Pietras, A. Östman, High interstitial fluid pressure—an obstacle in cancer therapy, Nat. Rev. Cancer 4 (2004) 806. [95] S.D. Conner, S.L. Schmid, Regulated portals of entry into the cell, Nature 422 (2003) 37. [96] D.A. Kuhn, D. Vanhecke, B. Michen, F. Blank, P. Gehr, A. Petri-Fink, et al., Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages, Beilstein J. Nanotechnol. 5 (2014) 1625 1636. [97] T.-G. Iversen, T. Skotland, K. Sandvig, Endocytosis and intracellular transport of nanoparticles: present knowledge and need for future studies, Nano Today 6 (2) (2011) 176 185. [98] (a) L. Josephson, C.-H. Tung, A. Moore, R. Weissleder, High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates, Bioconjug. Chem. 10 (2) (1999) 186 191. (b) M. Lewin, N. Carlesso, C.-H. Tung, et al., Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells, Nat. Biotechnol. 18 (2000) 410. [99] M. Dominska, D.M. Dykxhoorn, Breaking down the barriers: siRNA delivery and endosome escape, J. Cell Sci. 123 (8) (2010) 1183 1189. [100] J.Y. Wang, J. Chen, J. Yang, H. Wang, X. Shen, Y.M. Sun, et al., Effects of surface charges of gold nanoclusters on long-term in vivo biodistribution, toxicity, and cancer radiation therapy, Int. J. Nanomed. 11 (2016) 3475 3485. [101] F. Naz, V. Koul, A. Srivastava, Y.K. Gupta, A.K. Dinda, Biokinetics of ultrafine gold nanoparticles (AuNPs) relating to redistribution and urinary excretion: a long-term in vivo study, J. Drug Target. 24 (8) (2016) 720 729. [102] E. Sadauskas, G. Danscher, M. Stoltenberg, U. Vogel, A. Larsen, H. Wallin, Protracted elimination of gold nanoparticles from mouse liver, Nanomed.: Nanotechnol., Biol. Med. 5 (2) (2009) 162 169. [103] R. Goel, N. Shah, R. Visaria, G.F. Paciotti, J.C. Bischof, Biodistribution of TNF-α-coated gold nanoparticles in an in vivo model system, Nanomedicine (London, England) 4 (4) (2009) 401 410. [104] F. Muhammad, A. Wang, W. Qi, S. Zhang, G. Zhu, Intracellular antioxidants dissolve man-made antioxidant nanoparticles: using redox vulnerability of nanoceria to develop a responsive drug delivery system, ACS Appl. Mater. Interfaces 6 (21) (2014) 19424 19433. [105] P. Keselman, E.Y. Yu, X.Y. Zhou, P.W. Goodwill, P. Chandrasekharan, R.M. Ferguson, et al., Tracking short-term biodistribution and long-term clearance of SPIO tracers in magnetic particle imaging, Phys. Med. Biol. 62 (9) (2017) 3440 3453. [106] Q. He, Z. Zhang, F. Gao, Y. Li, J. Shi, In vivo biodistribution and urinary excretion of mesoporous silica nanoparticles: effects of particle size and PEGylation, Small 7 (2) (2011) 271 280. [107] S. Goy-López, J. Juárez, M. Alatorre-Meda, E. Casals, V.F. Puntes, P. Taboada, et al., Physicochemical characteristics of protein NP bioconjugates: the role of particle curvature and solution conditions on human serum albumin conformation and fibrillogenesis inhibition, Langmuir 28 (24) (2012) 9113 9126.

2 Biodistribution, Excretion, and Toxicity of Nanoparticles Yongfeng Zhao1, Deborah Sultan2, Yongjian Liu2 1

DEPARTM ENT O F CHEM IST RY , JACK SON STAT E UNIVERSITY, JACKSON, M S, UNITED STATES 2 MALLINCKRODT INSTITUTE OF RADIOL OGY, WASHINGTON UNIVERSITY, ST. LOUIS, M O, UNIT ED STATE S

2.1 Introduction The role of nanomaterials in the diagnosis and treatment of disease has been studied extensively [1 7]. There is much to consider with the introduction of nanomaterials into medicine—how will they be biologically distributed post administration? How will size, charge, shape, and surface functionalization affect in vivo pharmacokinetics? What toxicity concerns should warrant additional study? As an example, nanomaterials can be engineered to exhibit extended blood circulation due to evasion of clearance from the circulation system [8]. Nanoparticles are comparable to biomolecules, such as antibodies or DNA, but larger than small molecules. Thus, their inherent size allows circulating nanoparticles a chance to interact with cell surface biomolecules which would otherwise go unnoticed, yet they are large enough to be crafted with moieties for imaging and therapeutics. In addition, nanomaterials can be engineered with targeting agents that will seek out a specific tissue through the addition of a homing group, or by changing their shape and surface charges. These theranostic applications of nanoparticles, offering detection through imaging and treatment via targeted drug delivery, hold great promise in tackling some of the most troubling diseases. As a result, nanoparticles have great potential as a platform for the elusive “magic bullet,” killing targeted cells while leaving healthy cells unaffected. Important objectives for researchers in nanomedicine include optimizing biodistribution, fine tuning targeting for ideal signal-to-noise ratio, ensuring efficient clearance, and minimizing potential toxicities. This can be accomplished through adjusting the nanoparticle shape, size, charge, and surface coating. However, each of these variables must be carefully considered, as changing one will invariably alter in vivo behavior. For example, coating a polymeric nanoparticle with poly(ethylene glycol) (PEG) is a key tool for extending circulation times, allowing escape from detection by the mononuclear phagocytic system (MPS), which tags foreign bodies with opsonin proteins and ensures their elimination through the liver and spleen. However, a nanoparticle that has been PEGylated will have a different size and charge, thus altering its in vivo behavior. For clinical translation of nanoparticles, excretion Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00002-X © 2019 Elsevier Inc. All rights reserved.

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and toxicity present a major road block to further application. The goal of future efforts in nanomedicine must focus on the optimization of biodistribution to favor regions of interest while addressing efficient excretion from the body and minimizing toxicity. In this chapter, the biodistribution, excretion, and toxicity of nanoparticles will be summarized with a focus on translation and therapy. Various strategies for controlling biodistribution will be discussed. Guidelines for designing nanoparticles for targeted accumulation in interested regions while minimizing uptake by healthy organs for drug delivery or diagnosis will be outlined. Finally, nanoparticle toxicity will be reviewed with translation in mind. Most of the examples in this chapter will deal with inorganic nanoparticles. Their easily modifiable size, shape, and surface coatings allow straightforward study of the effect changes to these parameters have. Because of their intrinsic properties, they have well-documented roles in imaging (magnetic resonance imaging, Raman spectroscopy, photoacoustic imaging, and fluorescence) and therapy (photothermal therapy, magnetic thermal ablation), thus providing excellent examples of the effect of modifications on biodistribution, excretion, and toxicity.

2.2 Biodistribution Biodistribution plays a very important role in the evaluation of a nanoparticle’s diagnostic and therapeutic efficacy, biocompatibility, and toxicity. Commonly used tools for measuring biodistribution include radiolabels, near-infrared fluorescence, and inductively coupled plasma mass spectrometry (ICP-MS). Among these, radiolabeling techniques are widely used approaches for pharmacokinetic studies, especially for in vivo evaluation [9,10]. The advantage of a radiolabel is its sensitivity: only femtomols of nanoparticles are needed, a concentration far below that needed for saturation of nanoparticles binding to cell surface receptors, there is no tissue penetration limitation, and sample preparation can be very simple and straightforward. In addition, using an imaging modality like positron emission tomography (PET) or single-photon emission computed tomography allows in vivo imaging and biodistribution of nanoparticles to be conducted in real time.

2.2.1 Effect of Surface Material 2.2.1.1 PEGylation To a large degree, the biodistribution of nanomaterials is dictated by their surface chemistry, which will affect the extent and specificity of protein binding. In order to reach their intended target, nanoparticles must have sufficient circulation time. Naked circulating nanoparticles are quickly tagged for removal by a mechanism called opsonosis. In this process a protein called opsonin is attached to the surface of the nanoparticle, thus flagging it for removal by the immune system’s macrophages via reticuloendothelial cells (Fig. 2 1) [11 14]. The nanoparticles, which are viewed as foreign objects, end up mainly in the liver and spleen. It is suggested that the increased rate of hepatic uptake is mainly mediated by opsonization [15]. In general, the primary driving forces for opsonization of nanoparticles are

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FIGURE 2–1 Circulating opsonin proteins adsorb to the surface of nanoparticles, raising a flag of catching exogenous material for clearance. Macrophages in RES tissues, mainly those of the liver and spleen, recognize this flag and eliminate nanoparticles from circulation by phagocytosis. RES, reticuloendothelial system. Adapted from A.J. Cole, V.C. Yang, A.E. David, Cancer theranostics: the rise of targeted magnetic nanoparticles, Trends Biotechnol. 29 (7) (2011) 323 332. doi: 10.1016/j.tibtech.2011.03.001. PubMed PMID: PMC3210200 [17].

hydrophobic and electrostatic interactions, together with conformational changes and associated changes in entropy. As was mentioned previously, a surface coating of PEG is the classic method for the stealth shielding of nanoparticles and leads to greatly extended circulation times. Factors that will affect the circulation and partitioning of nanoparticles in vivo include PEG chain length, PEGylation surface density, and conformation [16]. A critical result of extended nanoparticle circulation is enhanced drug delivery to tumor sites. Traditional chemotherapeutic drugs are frequently administered in high doses and carry the burden of potentially lethal toxicity. The tunable pharmacokinetic behavior of nanoparticles in vivo elucidates an intriguing use as a drug carrier that can deliver cytotoxic drugs directly to the tumor while alleviating much of the damage to healthy cells. This delivery mechanism frequently relies on the tumor microenvironment, which consists of poorly regulated and irregularly shaped blood vasculature and a malfunctioning lymphatic drainage system. In such an environment drug-laden nanoparticles can deliver a payload by taking advantage of the so-called enhanced permeability and retention (EPR) effect [18]. It is important to note that while nanoparticle PEGylation increases circulation time it can also have a detrimental effect on cellular uptake by interfering with nanoparticle cell interactions [19]. Repeated injections of PEGylated particles are suspected to induce immune response and hypersensitivity, especially when an immunostimulatory agent is included [20]. This immune response results in subsequent injections being rapidly cleared via opsonosis, thus greatly diminishing the stealth characteristics sought with PEGylation. Issues such as these resulted in what is coined the “PEG Dilemma,” and led to researchers investigating alternative nanoparticle coatings [21]. Zwitterionic materials have emerged as an efficient alternative to reduce opsonin protein binding of nanomaterials [22]. Benefiting from the synergistic effects of zwitterionic and multivalent galactose polymers, drug-loaded nanoparticles were selectively internalized by cancer cells rather than normal tissue cells [23]. Gold surfaces modified with Cys-b exhibited prominent repellence against the nonspecific adsorption of proteins, bacteria, and fibroblast cells [24]. Care must be taken to thoroughly assess the effect of surface modifications on biodistribution, as some may have unintended consequences. For

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example, Muro et al. were surprised to find that coating nanoparticles with intercellular adhesion molecule I (ICAM-1) resulted in the cellular uptake of the nanoparticles. This was unexpected, as ICAM-1 is not known to trigger endocytosis [25]. Nanoparticles capped with novel zwitterionic disulfide ligands showed remarkable stability in saline media with salt concentrations as high as 3.0 M. Similarly, gold nanoparticles (AuNPs) did not precipitate out of solution when charged polyelectrolytes or biopolymers were added, indicating the absence of nonspecific interactions [26]. At a cellular level, by adjusting the hydrophobicity of zwitterionic coating on AuNPs, both their affinities to the cell membrane and the endocytosis kinetics were significantly, indicating the potential of zwitterionic ligands as a new surface coating material to further improve the biomedical applications of nanoparticles [27].

2.2.1.2 Charge The surface material of nanoparticles dictates the hydrophobic or hydrophilic properties as well as the charge. When introduced into a biological system, nanoparticles are immediately surrounded by proteins, resulting in a corona that will govern further interactions with other proteins and cells. The equilibrium kinetics of this process are affected by nanoparticle surface charge and must be taken into account when studying nanoparticle behavior in vivo [28]. An introduction of surface charge onto nanoparticles is typically reserved for specific instances to improve stability or prevent aggregation. However, this does shed light on an important consideration—the interplay between the negatively charged cell membrane and the positive or negative nanoparticle charge. Leroueil et al. used atomic force microscopy to show that cationic nanoparticles were able to disrupt and cross supported lipid bilayers regardless of size, shape, and flexibility. However, this disruption of the cellular membrane could generate pores in the protective layer, resulting in cellular toxicity caused by interrupted equilibrium of critical ions, proteins, and other macromolecules [29]. Neutral and anionic particles can still be adsorbed onto the cell membrane but were internalized at much lower levels [30]. Apart from cellular internalization, surface charge has a significant effect on biodistribution and ultimately the fate of the nanoparticle. Levchenko et al. prepared phosphatidylcholine/cholesterol liposomes with different charge status and different PEG length coatings (PEG750 or PEG5000) of approximately 200 nm in size and studied both in vitro behavior and in vivo tissue distribution in mice [31]. They demonstrated that the rate of clearance from the blood was significantly higher for negatively charged liposomes (potential B 40 mV) than for neutral liposomes (potential B10 mV). The negatively charged liposomes also show an increased rate of MPS uptake in the liver compared to neutral liposomes, indicating that phagocytic cells favor the uptake of negatively charged particles and, thus, increased the rate of clearance of particles from the blood. However, their studies also indicated that this charge effect could be counteracted based on the length and density of PEG chains used to coat the nanoparticle surface. In vivo studies demonstrated the complexity of these interactions, as the chemical composition of the charged lipid dictated the shielding effect of the PEG chains.

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In another study, in vivo evaluation of rhodamine-B-labeled chitosan nanoparticles showed charge-dependent opsonosis, with higher charged particles more attractive to macrophage uptake and ultimate deposition in the liver and spleen. Particles that were # 150 nm avoided opsonosis and instead underwent cellular internalization with greater efficiency than their larger counterparts. Surface charge played a smaller role in cellular internalization: either a weak negative charge or a strong positive charge led to increased uptake. Nanoparticle surface charge also played a part in circulation time: in this situation, it was the negatively charged particles that demonstrated extended circulation. On the other hand, once a target tumor site was reached it was the positively charged particles that more successfully left the circulation pool and targeted the tumor cells [32]. Jin et al. also demonstrated the effect of surface charge on cellular internalization [33]. They utilized lanthanidedoped upconversion nanoparticles (UCNPs) coated with polyvinylpyrrolidone (PVP), polyethylenimine (PEI), or polyacrylic acid (PAA) to generate UCNP-PVP, UCNP-PEI, and UCNP-PAA. The positively charged UCNP-PEI greatly enhanced cellular uptake in comparison with its neutral or negative counterparts, as shown by multiphoton confocal microscopy and ICP-MS measurements. Meanwhile, they found that cationic UCNP-PEI could be effectively internalized mainly through the clathrin endocytic mechanism, as revealed by colocalization, chemical, and genetic inhibitor studies. Sultan et al. studied the effect of charge on enabling gold nanoclusters to cross the blood brain barrier (BBB) following focused ultrasound, finding that the neutrally charged nanoclusters entered the brain with the greatest efficiency [34]. These experiments establish the complexity of the effect that not just charge, but also size and composition, have on biodistribution and clearance, demonstrating that these factors must be considered in concert when designing and testing nanoparticles. Much research has focused on the effect of positive, negative, or neutral charges on nanoparticle biodistribution. Han et al. compared the effect of a zwitterionic charge placement on cellular uptake of quantum dots (QDs) and found that zwitterionic QDs displaying positive charges in their outermost layer exhibit significant nonspecific accumulation to cultured cells and vessels in live mice, whereas zwitterionic QDs displaying negative charges in their outermost layer showed virtually no nonspecific adsorption [35]. Murthy et al. investigated the properties of zwitterionic AuNPs, specifically the hydrodynamic diameter shift caused by nonspecific binding of circulatory system proteins to their ultrasmall (5 nm) AuNPs. They noted that adherence of a single plasma protein may increase the AuNP size to greater than 6 nm, greatly reducing renal clearance. Through the construction of a binary mixed-charge monolayer on the surface of their AuNPs, they found negligible protein binding in undiluted fetal bovine serum by dynamic light scattering measurements [36]. The neutral property of PEG on the surface of nanoparticles has proved to be an effective method to improve blood circulation. The PEG density on the surface of nanoparticles affects not only circulation time but also the targeting capability. A study comparing packing densities of ligand-functionalized PEG onto nanoemulsions looked at αvβ3-integrin targeting efficiency with PEG density ranging from 5 to 50 mol.%. Consistent with the PEG density conformation models, both in vitro and in vivo studies demonstrate that low PEG surface density, at which the PEG chains are present in a mushroom configuration, is optimal for

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efficient and specific nanoparticle targeting. At higher densities the PEG chains form a brush layer on the particles, potentially hindering interaction of the targeting moiety with the ligand [15,37]. There are considerations beyond blood circulation when assessing in vivo nanoparticle behavior. A recent study demonstrated that surface charge also affects the suborgan distributions of ultrasmall (2 nm) AuNPs in the kidney, liver, and spleen of mice. Images of the kidney showed that positively charged nanoparticles accumulated extensively in the glomeruli, the initial stage in filtering for the nephron, suggesting that these nanoparticles may be filtered by the kidney at a different rate than the neutral or negatively charged nanoparticles. Both positively and negatively charged nanoparticles accumulated extensively in the red pulp of the spleen. In contrast, uncharged nanoparticles accumulated in the white pulp and marginal zone of the spleen to a greater extent than the positively or negatively charged nanoparticles. Moreover, these uncharged nanoparticles were more likely to be found associated with Kupffer cells in the liver. Positively charged nanoparticles accumulated extensively in liver hepatocytes, whereas negatively charged nanoparticles showed a broader distribution in the liver. Together these observations suggest that neutral nanoparticles having 2-nm cores may interact with the immune system to a greater extent than charged nanoparticles, highlighting the value of determining the suborgan distribution of nanomaterials for drug delivery and imaging applications [38].

2.2.2 Effect of Nanoparticle Size A critical consideration when designing a nanoparticle for optimal biodistribution, excretion, and toxicity is size. On the basis of physiological parameters such as blood circulation halflife, molecular targeting, and cellular uptake it is clear that, along with surface composition, particle size plays a significant factor and is key to achieving therapeutic efficacy. While generalizations can be made about optimal nanoparticle size, the biological system targeted by the nanoparticle is the ultimate deciding factor. The size of a nanoparticle will determine which clearance system will be utilized and the speed with which clearance occurs. When nanoparticles are administered by intravenous (i.v.) injection, those larger than 200 nm will be quickly filtered out of the bloodstream by the MPS system [39]. Nanoparticles smaller than 5 nm can pass through the kidney capillaries and are rapidly excreted by the renal system [40]. Nanoparticles that fall between 200 and 5 nm tend to have an extended blood circulation. An exception to this occurs in the case of tumor vasculature. While healthy blood vessels only allow small molecules to diffuse through, tumor lesions, because of their leaky blood vessels, allow larger nanoparticles to infiltrate and become trapped via the EPR effect (Fig. 2 2) [18,41,42]. Wang et al. studied the biodistribution of gold nanocages of varying sizes. Pharmacokinetic studies with femtomolar administration suggested that 30-nm nanocages had a greatly improved biodistribution profile as compared to 55-nm nanocages, together with higher blood retention and lower hepatic and splenic uptake in a murine EMT6 breast cancer model. The smaller cages also showed a significantly higher level of tumor uptake

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FIGURE 2–2 Schematic illustration of intravenously injected nanoparticle biodistribution showing accumulation in tumors due to the EPR effect, nanoparticle uptake by the RES system, and nanoparticle excretion by the kidney. EPR, Enhanced permeability and retention; RES, reticuloendothelial system. Adapted from G.M. Soliman, A. Sharma, D. Maysinger, A. Kakkar, Dendrimers and miktoarm polymers based multivalent nanocarriers for efficient and targeted drug delivery, Chem. Commun. 47 (34) (2011) 9572 9587. doi: 10.1039/C1CC11981H [43].

and a greater tumor-to-muscle ratio than the larger cages [44]. In a system using liposomes of varying sizes, however, 100-nm nanoparticles were found to have higher tumor uptake compared to their smaller counterparts. This difference in optimal size may rise from the nanoparticle composition as liposomes are flexible while inorganic nanoparticles are rigid [45]. Zhao et al. studied 10-nm AuNPs and observed high uptake in both tumor and the MPS system [46]. Using chelator free radiolabeling, they prepared 64Cu alloyed gold nanoclusters (64CuAuNCs) with different sizes for in vivo evaluation of pharmacokinetics, clearance, and PET imaging in a mouse prostate cancer model. Through PEGylation with 350 Da PEG, the 64 CuAuNCs-PEG350 with a hydrodynamic diameter of 4 nm exhibited optimal biodistribution and significant renal and hepatobiliary excretion. However, the nanocluster coated with PEG 1000 with a hydrodynamic diameter of 7 nm show longer blood circulation, significant uptake by MPS and limited renal clearance (Fig. 2 3). PET imaging showed low nonspecific tumor uptake, indicating its potential for active targeting of clinically relevant biomarkers in tumor and metastatic organs [47]. Hoshyar et al. compiled an excellent review article on the effect of nanoparticle size on in vivo pharmacokinetics and cellular interaction, which details many of the fine nuances of nanoparticle design for specific biological systems [48].

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FIGURE 2–3 Biodistribution of alloyed (A) 64CuAuNCs-PEG350 and (B) 64CuAuNCs-PEG1000 in wild-type C57BL/6 male mice at 1, 4, and 24 h post injection (n 5 4 per group). 64CuAuNCs, 64Cu alloyed gold nanocluster; PEG, poly (ethylene glycol). Adapted from Y. Zhao, D. Sultan, L. Detering, H. Luehmann, Y. Liu, Facile synthesis, pharmacokinetic and systemic clearance evaluation, and positron emission tomography cancer imaging of 64Cu-Au alloy nanoclusters, Nanoscale 6 (22) (2014) 13501 13509. doi: 10.1039/C4NR04569F.

2.2.3 Effect of Nanoparticle Shape One advantage of AuNPs is the variety of shapes in which they can be synthesized, including spheres, rods, disks, and cages. Wang et al. studied the shape effect on the biodistribution of gold nanomaterials by ICP-MS. They compared the in vitro and in vivo capabilities of Au nanohexapods as photothermal transducers for theranostic applications by benchmarking against those of Au nanorods and nanocages. While each Au nanostructure could absorb and convert near-infrared light into heat, Au nanohexapods exhibited the highest cellular uptake and the lowest cytotoxicity in vitro for both the as-prepared and PEGylated nanostructures. In vivo pharmacokinetics showed that the PEGylated Au nanohexapods had

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significant blood circulation and tumor accumulation in a mouse breast cancer model [49]. Radioactive Au nanostructures were synthesized with a similar size but four different shapes by directly incorporating 198Au into their crystal lattices. The radiolabeling integrity and stability associated with these nanostructures enabled quantitative analysis of biodistribution, tumor uptake, and intratumoral distribution using a murine EMT6 breast cancer model. Specifically, biodistribution was quantified by measuring the γ radiation from 198 Au, whereas both tumor uptake and intratumoral distribution were measured by detecting the β1 emission for Cerenkov radiation and autoradiography. Of the four shapes, the 198Au-incorporated nanospheres showed the best blood circulation, the lowest MPS clearance, and the highest overall tumor uptake relative to nanodisks, nanorods, and nanocages. Interestingly, nanorods and nanocages reached the cores of the tumors, whereas nanospheres and nanodisks were only observed on the surfaces of the tumors, suggesting the unique positions of Au nanorods and nanocages for photothermal cancer treatment [50]. Aspect ratio (AR) has a profound impact on the behavior of the nanoparticles in vivo and in vitro [51]. PEGylated nanorods with the lowest AR (3.5) achieved the most efficient passive tumor homing behavior because they could diffuse easily, whereas Arginylglycylaspartic acid (RGD)-labeled particles with a medium AR (7) were more efficient at tumor targeting because this requires a balance between infusibility and ligand receptor interactions. The in vivo behavior of nanoparticles can therefore be tailored to control biodistribution, longevity, and tumor penetration by modulating a single parameter: the AR of the nanocarrier. Li et al. investigated the effect of ARs ranging from 1.5 to 5 with spherical, short rod, and long rod mesoporous silica nanoparticles (MSNs). Biodistribution studies following intragastric administration showed detectable MSN levels in the liver, lung, spleen, and kidney 2 h post administration, indicating their entry into the circulatory system. The rod-shaped MSNs had a higher uptake in the liver and lung, while the sphere-shaped particles had increased uptake in the spleen. Liver uptake of the spherical MSNs increased continuously to the 72-h time point, while the rod-shaped MSNs followed an opposite trend. In examining toxicity, they found no histological abnormality in the small intestine, heart, liver, spleen, or lung following oral administration. Kidney damage was observed in all of the study groups with the greatest damage coming from the spherical MSNs and only slight hemorrhage from their rod-shaped counterparts [9]. Guo et al. studied the effect of both size and shape on in vivo biodistribution using RNA nanoparticles tagged with Alexa Fluor 647 and a nude mouse KB tumor model. In these experiments, RNA nanoparticles of comparable sizes but varying shape (triangle, square, and pentagon) were compared with those of similar shapes (square) but sizes of 5, 10, or 20 nm. They found that circulation time increased as size increased from 5 to 20 nm, the common size range of therapeutic RNA nanoparticles. Liver, spleen, and kidney accumulation was greatest for the 20-nm RNA nanoparticle. This size also had the greatest tumor accumulation, but the signal-to-noise ratio for the tumor was best in the 5- and 10-nm sizes. When observing the effect of nanoparticle shape, the pentagon showed the greatest tumor accumulation though all three shapes had signal [52].

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2.2.4 Effect of Nanoparticle Rigidity Recently, greater degrees of nanoparticle flexibility were hypothesized to improve the binding ability of particles on the cell surface [53]. In an interesting study, zwitterionic nanogels of varying stiffness were prepared by tuning their crosslinking densities and reactant contents. In vivo studies of these nanogels show that softer nanogels passed through physiological barriers, especially the splenic filtration, more easily than their stiffer counterparts, consequently leading to longer circulation half-life and lower splenic accumulation (Fig. 2 4) [54]. To evaluate the effects of the rigidity of the polymeric core on the in vivo biodistribution, Wooley et al. synthesized nanoparticles with similar physiochemical properties (size, c. 20 nm; charge c. 25 mV) possessing a low glass transition temperature (Tg) with a fluid-like poly(methyl acrylate) (PMA) core or a high Tg with a glassy poly(styrene) (PS) core. The results show that high-Tg PS core nanoparticles exhibited a significantly higher blood residence time compared to the low-Tg PMA nanoparticles. The low-Tg core was expected to provide greater flexibility and an increased number of surface interactions of the nanoparticles with tissues and the biological environment. However, it was not clear if the relative rigidity or other physicochemical properties of the polymers (hydrophobicity) affected the blood residence time [55].

2.2.5 Effect of Administration Route Another consideration in the effective use of nanoparticles for imaging and therapy is the administration route. The organ system to which a nanoparticle is introduced, be it the digestive system via oral administration or the circulatory system via injection, will invariably result in different distribution pathways in vivo. This will affect clearance as demonstrated by Yu et al. They found that PEI modified NaYF4:Yb, ER UCNP (PEI@UCNPs) injected by intraperitoneal (i.p.) had high spleen accumulation and slow distribution to other organs. Following oral administration, clearance was mainly through the intestinal tract [56]. In a study examining the toxicological effects of colloidal AuNPs, high concentrations administered either orally or by i.p. showed the greatest toxicity resulting in weight loss, decreased

FIGURE 2–4 Interactions of hard particles and soft particles with slits smaller than their size; the hard particles are trapped by the slit, while the soft particles can deform and pass through the slit. Adapted from L. Zhang, Z. Cao, Y. Li, J.-R. Ella-Menye, T. Bai, S. Jiang, Softer zwitterionic nanogels for longer circulation and lower splenic accumulation, ACS Nano 6 (8) (2012) 6681 6686. doi: 10.1021/nn301159a.

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spleen index, and decreased red blood cell counts in a mouse model. Conversely, i.v. injection via tail vein showed decreased toxicity. Low concentrations of the AuNPs did not show any significant toxicity [57]. Before nanoparticles enter into the systemic circulation, they need to overcome different biological barriers such as mucosa. Xie et al. study the impact of intratumoral injection on the biodistribution of gold nanoshells for phototherapy. They found a higher intratumoral retention of nanoshells and a lower concentration in other healthy tissues, suggesting that intratumoral administration is potentially a better approach for nanoshell-based photothermal therapy [58]. In a study of photoluminescent carbon-based C-dot nanoparticles, the effect of three injection routes (i.v., subcutaneous, and intramuscular) on in vivo circulation, clearance, and tumor accumulation was explored. Initially, i.v. administration led to blood levels much greater than either subcutaneous or intramuscular routes. However, the i.v. injected C-dot-ZW8000 cleared rapidly from the blood pool while both subcutaneous- and intramuscular-administered C-dots accumulated in blood over time. As a result, at 60 min post injection (p.i.) blood levels were slightly higher for subcutaneous- and intramuscular-administered C-dots. Interestingly, the biodistribution profile of C-dots was similar regardless of injection route (Fig. 2 5). Clearance was predominantly via the kidneys with little signal in any organ system 24 h p.i. [59]. In an examination of tumor uptake, C-dot-ZW8000 particles were injected into athymic nude mice with murine squamous cell carcinoma-7 tumors following one of the three administration routes. In each case tumor uptake was easily observed at 2 h p.i. and remained high over time. Both i.v. and subcutaneous injection resulted in greater tumor fluorescence than intramuscular, and the subcutaneous had the greatest signal at later time points, presumably due to its slower blood clearance.

FIGURE 2–5 Quantification of the biodistribution of 64Cu labeled C-dots via three injection routes at 1 h (A) and 24 h (B) time points. Adapted from X. Huang, F. Zhang, L. Zhu, K.Y. Choi, N. Guo, J. Guo, et al., The effect of injection routes on the biodistribution, clearance and tumor uptake of carbon dots, ACS Nano 7 (7) (2013) 5684 5693. doi: 10.1021/nn401911k. PubMed PMID: PMC3725601.

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2.3 Excretion Upon introduction of nanoparticles into a biological system, circulation results in clearance from the blood and entry into different tissues. For biomedical applications, FDA regulations favor nanoparticle excretion within a certain window of time [40]. There are two major excretion routes for nanoparticles: renal (urine) and hepatic (bile to feces). Biological clearance is classified into three categories: (1) disintegration of nanoparticles by protein adsorption, (2) opsonization-mediated nanoparticle removal by immune cells, and (3) filtration by organs with fenestrated vasculature (Fig. 2 6) [60].

(A)

(B)

Nanoparticle drainage route

Kidney

Renal clearance Tubule cells

Lungs

Pulmulary clearance Alveolus Alveolar macrophage

Tubule lumen

Particle size < 5 nm

Hepatic clearance

Particle size > 100 nm

RES/MPS clearance

Spleen

Liver (C)

(D)

Sinusoidal lumen Disse space

Particle size > 20 to < 100 nm

Sinusoidal space

Kupffer's cell Stellate cell

Hepatocytes

Particle size < 20 nm

Particle size > 20 nm

Macrophage

FIGURE 2–6 Drainage of nanoparticles through different organs according to their properties: The kidney is the most traditional drainage system eliminating the particles ,5 nm (A) and the particles ,5 nm are easily cleared from the blood. Although the lungs (B) are not a conventional eliminatory system, they can help in filtering aerosolic nanoparticle with sizes .100 nm. A second level of elimination is done through the liver (C), where particles of size 20 100 nm can pass through. The larger particles ( . 200 nm) which are not eliminated through the kidney or liver are eventually eliminated by the MPS, for example, lymph node and spleen (D). Adapted from T. Bose, D. Latawiec, P.P. Mondal, S. Mandal, Overview of nano-drugs characteristics for clinical application: the journey from the entry to the exit point, J. Nanopart. Res. 16 (8) (2014) 2527. doi: 10.1007/s11051-014-2527-7 [61].

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2.3.1 Mononuclear Phagocytic System Clearance The dominant clearance pathway for nanoparticles is the MPS, resulting in liver and spleen accumulation. In 10-nm AuNPs doped with 64Cu, liver and spleen uptake can be up to 100% of injected dose (Fig. 2 7) [46]. Uptake by MPS is greater if the hydrodynamic diameter of the nanoparticle is greater than 10 nm. Smaller nanoparticles tend to be cleared via the renal system. In addition to large size, adsorption of opsonins onto nanoparticles initiates rapid recognition by receptors on the cell surface of macrophages in the MPS (such as Kupffer cells in the liver) and subsequent internalization. To study the mechanism of nanomaterial clearance by the liver, Tsoi et al. found that nanomaterial velocity reduces 1000-fold as they enter and traverse the liver, leading to 7.5 times more nanomaterial interaction with hepatic cells relative to peripheral cells (Fig. 2 8) [62]. To decrease nanoparticles’ MPS clearance, the typical approaches are the reduction of nanoparticle size or surface modifications, such as PEGylation [63]. PEGylation will decrease the speed of protein attachment to nanoparticles. Eventually, larger nanoparticles will be cleared from the blood circulation system by MPS regardless of surface modifications of various types of nonrenally clearable inorganic NPs with or without a PEG-coated surface (gold nanomaterials, QDs, iron oxide NPs, lanthanide upconversion NPs, silica NPs, and carbon

FIGURE 2–7 Biodistribution of alloyed 64CuAuNPs in female BALB/c mice via tail vein administration at 1, 24, and 48 h post injection (n 5 4/group). Adapted from Y. Zhao, D. Sultan, L. Detering, S. Cho, G. Sun, R. Pierce, et al., Copper-64-alloyed gold nanoparticles for cancer imaging: improved radiolabel stability and diagnostic accuracy, Angew. Chem. Int. Ed. 53 (1) (2014) 156 159. doi: 10.1002/anie.201308494.

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FIGURE 2–8 Mechanism of nanomaterial transport in the liver. Adapted from K.M. Tsoi, S.A. MacParland, X.-Z. Ma, V.N. Spetzler, J. Echeverri, B. Ouyang, et al., Mechanism of hard-nanomaterial clearance by the liver, Nat. Mater. 15 (2016) 1212. doi: 10.1038/nmat4718. https://www.nature.com/articles/nmat4718#supplementary-information.

nanomaterials). There is no significant difference in the liver uptake between the PEGylated NPs (34.1% 6 21.8% ID/g, N 5 29) and non-PEGylated NPs (28.1% 6 15.3% ID/g, N 5 12) (P..05) 24 h p.i. [64]. Nanoparticles composed of inorganic materials are stable and thus difficult to break down by lysosomal enzymes. As a result, they can remain in the body for a long time—up to several years [65,66]. For example, 24 h after i.v. injection of 40-nm citrate-coated AuNPs, transmission electron microscopy (TEM) analysis showed accumulation in nearly all Kupffer cells in the liver and in lysosome/endosome-like vesicles. Six months later, ICP-MS analysis showed this accumulation decreased just 9% [67]. According to a pilot study in nonhuman primates, more than 90% of the injected dose of CdSe/CdS/ZnS QDs remained in the major organs, including liver and spleen, at 90 days post i.v. injection [68]. CdSe ZnS QDs with ligand-stabilized surface were retained in the body for at least 2 years; some remaining intact and showing fluorescence [69]. For other NPs that can decompose inside macrophages, such as silica NPs [70] and iron oxide NPs [71], bile excretion was observed after MPS uptake but elimination was very slow (weeks to months) compared with renal clearance of small molecular probes (hours to days). For iron oxide nanoparticles, the in vivo fate of PEGylated monodisperse nanocrystals and the resulting iron, phospholipid, and oleic acid biodegradation products may influence the cellular environments in the organs and blood, resulting in safety concerns.

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2.3.2 Renal Clearance To address concerns over the extended residence times seen in MPS, reduced nanoparticle size resulting in renal clearance is a viable route [72]. When the size of nanoparticles is less than 5.5 nm, they can be cleared from the blood circulation efficiently [73]. Bawendi et al. found that varying PEG chain length resulted in major changes in organ/tissue-selective biodistribution and clearance [74]. This result is further confirmed by chelator free radioisotope labeled AuNPs [47,75]. Varying PEG lengths on the surface of these gold nanoclusters led to either renal clearance (PEG 350) or MPS clearance (PEG1000) (Fig. 2 9). Zwitterionic or neutral organic coatings on QDs prevented adsorption of serum proteins, which otherwise increased hydrodynamic diameter by over 15 nm and prevented renal excretion. A final size smaller than 5.5 nm resulted in rapid and efficient urinary excretion, and elimination of the QDs from the body [40]. Yu et al. studied the effect of surface coatings on the clearance of renal clearable gold nanoclusters with PEG and zwitterion surfaces [64]. Although the tumor targeting and tumor-to-blood ratio were higher for PEGylated nanoclusters, the renal clearance was almost identical. These results indicated that size is a major factor in determining the clearance route. It is noteworthy that some research reported silicon nanoparticles with size greater than 50 nm could also be cleared through the renal system [76 78], reasonably due to the high hemoconcentration of silicon nanoparticles which resulted in a high excretory rate through urine in the initial state. These results were confirmed by measurement with ICP-MS [79], fluorescence and TEM [77], and ICP-MS and TEM [78].

FIGURE 2–9 Clearance profiles of (A) 64CuAuNCs-PEG350 and (B) 64CuAuNCs-PEG1000 in wild-type C57BL/6 male mice after intravenous injection. 64CuAuNCs, 64Cu alloyed gold nanocluster; PEG, poly(ethylene glycol). Adapted from Y. Zhao, D. Sultan, L. Detering, H. Luehmann, Y. Liu, Facile synthesis, pharmacokinetic and systemic clearance evaluation, and positron emission tomography cancer imaging of 64Cu-Au alloy nanoclusters, Nanoscale 6 (22) (2014) 13501 13509. doi: 10.1039/C4NR04569F.

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2.4 Toxicity Nanoparticle toxicity can arise from a number of factors. Slowly clearing nanoparticles may pose toxicity concerns as they interact with biological systems, potentially forming new species. While gold and iron oxide nanoparticles are composed of materials that are considered biocompatible, any substance can be toxic at high concentrations. In some instances it is the composition of the nanoparticle itself that causes toxicity. Thus performing a thorough toxicity study is critical for any nanoparticle that will be considered for in vivo administration. As mentioned previously, the administration route can affect nanoparticle toxicity. The following examples focus mainly on i.v. administration [57]. When a nanoparticle is composed of two or more materials, toxicity studies can begin by examining the toxicity of each individual component. However, a recent study by Hutchison et al. showed a synergistic effect between nanoparticles and Polysorbate 20, a surfactant commonly used in nanoparticle applications to aid dispersion and enhance performance. Although the AuNPs and surfactant individually show minimal toxicity in zebrafish, when combined the toxicity increased dramatically. These results demonstrate the importance of performing toxicity studies not just on the nanoparticle of interest but also the matrix with which it will be delivered [80].

2.4.1 Liver Toxicity The liver is the most common site for clearance of nanoparticles because this organ is where the nanoparticle will be deposited, digested, and disposed of, hence liver toxicity is typically the first to be considered. Of various nanoparticles, AuNPs are widely investigated because of their broad preclinical and clinical applications. At low concentrations, AuNPs are considered to be safe due to their inert chemistry. Still, there have been reports on AuNP toxicity, which is typically a result of the nanoparticle’s physical dimension, surface chemistry, and shape. Huang et al. carried out a study to examine the possible toxicity of AuNPs. Naked AuNPs ranging from 3 to 100 nm were injected intraperitoneally into BALB/ c mice at a dose of 8 mg/kg/week. At sizes of 3, 5, 50, and 100 nm the AuNPs did not show harmful effects; however, AuNPs ranging from 8 to 37 nm induced severe sickness in mice. Pathological examination of the major organs of mice in the diseased groups indicated an increase of Kupffer cells in the liver, loss of structural integrity in the lungs, and diffusion of white pulp in the spleen. These signs of toxicity were associated with the presence of AuNPs at the diseased sites [81]. Despite improved cytocompatibility conferred by biological surface coatings, the primary mechanism of liver toxicity is thought to arise from acute inflammatory changes and subsequent apoptosis [82,83]. It has been thought that the toxic effects of iron oxide NPs are due to the excessive production of radical oxygen species (ROS). These generated ROS further elicit DNA damage and lipid peroxidation [84]. Much more effort is required to develop magnetic iron oxide NPs with improved biocompatible surface engineering to achieve minimal toxicity, for various applications in biomedicine. A study conducted by Zhou et al. suggested that excess iron resulting from a high dose of iron oxide NPs might be a risk factor for cirrhosis because of

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the marked impacts of elevated lipid metabolism, disruption of iron homeostasis, and potential aggravated loss of liver functions [85]. For nanoparticles containing heavy metals, the toxicity of the metals themselves raises extra concerns. A study by Bhatia et al. suggests that CdSe-core QDs were indeed acutely toxic under certain conditions. They found that the cytotoxicity of CdQDs was modulated by processing parameters during synthesis, exposure to ultraviolet light, and surface coatings. Their data further suggest that cytotoxicity correlates with the liberation of free Cd21 ions due to deterioration of the CdSe lattice. When appropriately coated, CdSe-core QDs were rendered nontoxic and used to track cell migration and reorganization in vitro [86]. In another study, Clift et al. carried out a series of experiments assessing additional surface coatings to cope with Cd-based QD cytotoxicities. CdTe/CdSe cored QDs with a ZnS shell were covered with organic, carboxylated (COOH), amino (NH2), or PEG coatings. Cytotoxicity was tested on exposure to each type by measurement of macrophage cell viability and layered double hydroxide release. All of the studied QDs were shown to induce significant cytotoxicity after 48 h and coating materials as well as liberated Cd ions were suggested to be the causative agents. It is likely that a breakdown of physically labile surface material resulted in ion liberation and subsequent toxicity [87].

2.4.2 Kidney Toxicity In vivo studies have demonstrated that nanoparticles exhibit potential adverse nephrotoxicity at both the tubular and glomerular levels [88]. For nanoparticles smaller than 6 nm, the kidney has been reported as a target organ for nanoparticles. As an example, nephrotoxicity is observed when CdS nanoparticles are administrated through gavage [89]. The possible reason is that cadmium can generate ROS as well as bind with cysteine residues in proteins. Feng et al. examined commercially available iron oxide NPs with either 10 or 30 nm core size and coated with either PEG (SMG-10, SMG-30) or PEI (SEI-10) to see variations in cellular uptake, biodistribution, clearance, and toxicity. SEI-10 showed greater cellular uptake by both macrophages and cancer cells in vitro but also demonstrated greater dose-dependent toxicity. For in vivo evaluation, the iron oxide NPs were injected into SKOV-3 tumor-bearing mice. Tumor accumulation via the EPR effect was ranked SMG-10 . SMG-30 . SEI-10. The negatively charged SEI-10 showed high kidney uptake, whereas the PEG-coated iron oxide NPs were mainly cleared in the liver and spleen. Mice injected with SEI-10 were found to have a maximum tolerated dose of 1.5 mg/kg, while no mortality was observed in mice injected with either SMG-10 or SMG-30 at doses up to 5 mg/kg [90]. The smallest nanoparticles are typically cleared by the renal system. Engineered QDs coated with a neutral or zwitterionic layer evaded adsorption of serum proteins, retained their small size (,6 nm), and were efficiently cleared by the kidney by crossing a unique multiple-layer structure of glomeruli. Kidney toxicity was low as there was little interaction between the renal tissues and nanoparticles. Those without a protective layer grew in size by over 15 nm and were retained in the liver and spleen [91]. However, the correlation between size and renal clearance has its limits. In a recent study, Zheng et al. reported that in the sub-nm range, AuNCs with a diameter around 1 nm were retained in the kidneys. By further

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reducing the number of Au atoms in the AuNCs renal retention increased by four to nine times. They found that at these ultrasmall sizes AuNCs were readily trapped by the glomerular glycocalyx of the glomerulus (Fig. 2 10). How (or if) these nanoparticle will be cleared is a question needing additional investigation [92].

FIGURE 2–10 Renal clearance of different-sized gold nanoclusters and schematic diagram of the glomerular filtration membrane. (A) Whole-body X-ray images of the mice after being intravenously injected with Au10 11, Au18 and Au25, respectively at 40 min post injection. While all three different AuNCs were cleared through the kidneys into the bladder, the smallest Au10 11 has much longer kidney retention than Au18, which in turn has longer kidney retention than Au25 even though there is only a 7-atom difference among these three AuNCs. (B), The X-ray intensity bladder to kidney ratios of Au10 11, Au18 and Au25 at 40 min post injection, clearly showing that more Au10 11 and Au18 were retained in the kidneys than Au25.  means P , 0.05 based on One-way ANOVA. (n 5 3 for Au10 11, Au25, n 5 4 for Au18) (C), Renal clearance efficiency of Au10 11, Au15, Au18 and Au25 in 0 2 h and 2 24 h after intravenous injection (n 5 3 for Au10 11, Au15, Au25, n 5 6 for Au18). (D), The renal clearance efficiencies of Au10 11, Au15, Au18 and Au25, 1.7 nm (Au201), 2.5 nm (Au640) and 6 nm (Au8856) GS-AuNPs 24h post injection over the number of gold atoms. Below Au25, the renal clearance efficiency exponentially decreased with the decrease of the number of gold atoms in the NPs. (E), Glomerulus12, an important component of renal filtration, is composed of kidney blood vessel, glomerular filtration membrane and Bowman’s space. The glomerular filtration membrane is composed of multiple layers: endothelial glycocalyx, endothelial cell, glomerular basement membrane (GBM) and podocyte. Podocytes are covered by 200 nm glycocalyx. Generally, the fenestration between endothelial cells is 70 90 nm; GBM junction is 2 8 nm; the sizes of podocyte slits are in the range of 4 11 nm. Combination of these layers, the size threshold for kidney filtration is B6 nm: NPs or proteins with a hydrodynamic diameter (HD) , 6 nm can pass through glomerular filtration membrane readily while it is difficult for the large ones to cross through it. Adapted from B. Du, X. Jiang, A. Das, Q. Zhou, M. Yu, R. Jin, et al., Glomerular barrier behaves as an atomically precise bandpass filter in a sub-nanometre regime, Nat. Nanotechnol. 12 (2017) 1096. doi: 10.1038/nnano.2017.170. https://www.nature.com/articles/nnano.2017.170#supplementary-information.

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2.4.3 Heart Toxicity Clearance routes are the obvious first place to look for signs of nanoparticle toxicity. However, critical organ systems must also be evaluated for any nanoparticle eyed for translation. Li et al. noted that AuNPs, while widely used, had not been evaluated for cardiotoxicity. For 2 weeks they performed daily injection in mice of PEG-coated AuNPs of either 10, 30, or 50 nm size. The mice were sacrificed 2, 4, or 12 weeks following the first injection. The only signs of cardiac distress were seen at 2 weeks in mice injected with 10-nm AuNPs. In this group, symptoms included increases in left ventricular end-diastolic inner dimension, left ventricular mass, and heart weight/body weight ratio. Interestingly, none of these symptoms was present in the same group at 4 or 12 weeks, indicating that these were reversible conditions [93]. In another study looking at repeated dosing of AuNPs in rats, 10-, 20-, and 50-nm AuNPs were injected for 3 or 7 days. Animals injected with AuNPs of 10 or 20 nm exhibited congested heart muscle, dilated blood vessels, scattered and extravasations of red blood cells, and a dense focus of inflammatory cells, while the rats injected with 50-nm AuNPs showed none of these symptoms [94]. In a study on the effect of silver nanoparticles (AgNPs) on cardiac electrophysiology, it was shown that AgNPs exert rapid toxic effects on myocardial electrophysiology and induced concentration-dependent symptoms in mice, including depolarized resting potential, diminished action potential, loss of excitability in cardiac musculature, and lethal bradyarrhythmias. These acute toxicities were not present in mice treated with Ag1 ions, indicating toxicity was from the AgNPs and not exposure to silver [95].

2.4.4 Brain Toxicity Damage to the brain and central nervous system is of great concern for nanomaterial applications. Fortunately, the extremely proficient BBB protects the brain from the majority of molecules circulating in the blood from entry. Because of this effectiveness, most research has focused on ways to cross the BBB for delivery of drugs to the brain [96,97]. Though there is no direct route for inhaled nanoparticles to translocate to the brain, passage through blood, the lymph system, olfactory mucosa, and cerebrospinal fluid is possible. These pathways have been established but show low efficiency [98]. As research into the use of nanomedicine for treating deadly diseases of the brain advances, careful consideration must be given to the potential of nanotoxicity.

2.4.5 Lung Toxicity The lungs are unique when it comes to potential nanoparticle toxicity. Due to the size of NPs, inhalation is the most direct path of entry to the pulmonary system. In addition, the closely synched pulmonary and cardiac systems result in pulmonary exposure to any NPs in the circulatory system as well. Exposure may be intentional, as in the case of targeting or therapeutic nanoparticles, or unintentional, via inhalation or dermal exposure as a result of the increasing number of industrial nanoparticle applications. As has been discussed with other organ systems, toxicity will be dependent on size, surface charge, composition, shape, and method of administration.

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Therapeutic uses of nanoparticles for lung applications typically focus on detection and treatment. Most of these applications still rely on administration through the bloodstream with moieties targeting the pulmonary system. These nanoparticles are typically cleared through the MPS system and thus do not proffer lung toxicity specifically. Carlson et al. looked at the effect of size and concentration on toxicity of AgNPs. Alveolar macrophages were exposed in vitro to either Ag-15 nm, Ag-30 nm, or Ag-55 nm in concentrations ranging from 10 to 75 μg/mL and evaluated for changes in cell morphology, viability, ROS, glutathione concentration, and cytokine/chemokine concentration. They found all three sizes of AgNPs were internalized into the macrophages after 24 h and demonstrated sizeand concentration-dependent toxicity, with the Ag-15 nm particle being the most damaging to cells. They suggested that cytotoxicity was likely facilitated by increasing ROS species and cytokine (monocyte chemotactic protein-1) levels [99]. In an effort to provide a more standardized testing method for NP lung toxicity, Lanone et al. evaluated 24 nanoparticles with similar size and shape but varying composition using two human pulmonary cell lines, A549 and THP-1. Toxicity was determined at 3 and 24 h post exposure with both 3-(4,5-dimethylthiazo(-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red assays. Copper and zinc NPs showed the highest toxicity, with TC50 values below 15 μg/mL, followed by titanium, aluminum, cerium, and zirconium NPs [100]. Commercially available copper oxide NPs were administered via intratracheal instillation in a C57BL/6 murine lung model. Dose-dependent infiltration of inflammatory cells and lymphocyte aggregation were seen at 7 days post instillation and worsened thereafter. Granulomatous inflammation, lung fibrosis, increased ROS, and epithelial cell apoptosis were also noted [101].

2.5 Challenges and Perspectives The vast field of growing knowledge in nanoparticles has demonstrated the promising applications of nanomedicine to the future of health care. Going forward, consideration of biodistribution, clearance, and toxicity must guide the design of nanoparticles for translational applications. Challenges currently existing need to be addressed: 1. Although nanoparticles have demonstrated an EPR effect, further improvement of tumor targeting is in great need. The low delivery efficiency is a major limitation of nanoparticles for translational applications [42]. 2. Because of their sizes, the majority of nanoparticles will not be cleared through the renal system. Liver and spleen uptake are the dominant clearance modes for most nanoparticles. Long-term toxicity must be evaluated in these instances. 3. The large-scale synthesis of uniform nanoparticles with controlled surface properties is in high demand. The size and surface coating of nanoparticles profoundly affect biodistribution, clearance, and toxicity. Streamlining nanoparticle synthesis is crucial for standardizing results across studies.

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Several nanoparticle-based drugs have been tested in clinical trials based on the EPR effect. It is not clear if the EPR effect applies to small tumor lesions or sites of metastasis. Future studies need to focus on improving the blood circulation half-life and pharmacokinetics of nanomaterials to reduce uptake by liver and spleen, testing of these nanostructures in human-derived tumor models, and adding molecular targeting ligands to increase tumor uptake for better use of the therapeutic capability. The pharmacokinetics and biodistribution of nanoparticles are dictated by their surface modifications. Thus the proper surface materials must be utilized for optimal in vivo behavior. The field of nanotechnology holds great promise for the diagnosis and treatment of human diseases. However, the size and charge of most nanoparticles preclude their efficient clearance from the body as intact nanoparticles. Without such clearance or biodegradation into biologically benign components, toxicity is potentially amplified and radiological imaging is hindered. For future applications, biodegradable nanoparticles will significantly advance the field forward from preclinical to clinical applications. In order to reduce nanoparticle toxicity, the development of renally clearable nanoparticles will be critical. The biological microenvironment also affects biodistribution, clearance, and toxicity. The physicochemical properties of nanoparticles, such as size, shape, surface charge, surface chemistry (PEGylation, ligand conjugation), and composition affect the pharmacokinetics, biodistribution, intratumoral penetration, and tumor bioavailability. On the other hand, tumor biology (blood flow, perfusion, heterogeneity, permeability, interstitial fluid pressure, and stroma content) and patient characteristics (age, gender, tumor type, tumor location, body composition, and prior treatments) also have an impact on drug delivery by nanoparticles. It is now believed that both nanoparticles and the tumor microenvironment have to be optimized or adjusted for optimal delivery [102]. When designing nanoparticles, a clear focus of the biological system being addressed must be foremost in the process.

References [1] Y. Min, J.M. Caster, M.J. Eblan, A.Z. Wang, Clinical translation of nanomedicine, Chem. Rev. 115 (19) (2015) 11147 11190. Available from: https://doi.org/10.1021/acs.chemrev.5b00116. PubMed PMID: 26088284; PMCID: PMC4607605. [2] X. Yang, M. Yang, B. Pang, M. Vara, Y. Xia, Gold nanomaterials at work in biomedicine, Chem. Rev. 115 (19) (2015) 10410 10488. Available from: https://doi.org/10.1021/acs.chemrev.5b00193. PubMed PMID: 26293344. [3] M. Elsabahy, K.L. Wooley, Design of polymeric nanoparticles for biomedical delivery applications, Chem. Soc. Rev. 41 (7) (2012) 2545 2561. Available from: https://doi.org/10.1039/c2cs15327k. PubMed PMID: 22334259; PMCID: PMC3299918. [4] M. Elsabahy, G.S. Heo, S.M. Lim, G. Sun, K.L. Wooley, Polymeric nanostructures for imaging and therapy, Chem. Rev. 115 (19) (2015) 10967 11011. Available from: https://doi.org/10.1021/acs.chemrev.5b00135. PubMed PMID: 26463640; PMCID: PMC4610256. [5] D. Furtado, M. Bjornmalm, S. Ayton, A.I. Bush, K. Kempe, F. Caruso, Overcoming the blood-brain barrier: the role of nanomaterials in treating neurological diseases, Adv. Mater. 30 (2018) e1801362. Available from: https://doi.org/10.1002/adma.201801362. PubMed PMID: 30066406.

48

THERANOSTIC BIONANOMATERIALS

[6] D. Kim, K. Shin, S.G. Kwon, T. Hyeon, Synthesis and biomedical applications of multifunctional nanoparticles, Adv. Mater. 30 (2018) e1802309. Available from: https://doi.org/10.1002/adma.201802309. PubMed PMID: 30133009. [7] E.B. Ehlerding, P. Grodzinski, W. Cai, C.H. Liu, Big potential from small agents: nanoparticles for imaging-based companion diagnostics, ACS Nano 12 (2018) 2106 2121. Available from: https://doi.org/ 10.1021/acsnano.7b07252. [8] S.-D. Li, L. Huang, Pharmacokinetics and biodistribution of nanoparticles, Mol. Pharmaceutics 5 (4) (2008) 496 504. Available from: https://doi.org/10.1021/mp800049w. [9] C. Cilliers, I. Nessler, N. Christodolu, G.M. Thurber, Tracking antibody distribution with near-infrared fluorescent dyes: impact of dye structure and degree of labeling on plasma clearance, Mol. Pharmaceutics 14 (5) (2017) 1623 1633. Available from: https://doi.org/10.1021/acs.molpharmaceut.6b01091. [10] Y. Liu, M.J. Welch, Nanoparticles labeled with positron emitting nuclides: advantages, methods, and applications, Bioconjugate Chem. 23 (4) (2012) 671 682. Available from: https://doi.org/10.1021/ bc200264c. [11] P. Aggarwal, J.B. Hall, C.B. McLeland, M.A. Dobrovolskaia, S.E. McNeil, Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy, Adv. Drug Delivery Rev. 61 (6) (2009) 428 437. Available from: https://doi.org/10.1016/j.addr.2009.03.009. PubMed PMID: PMC3683962. [12] V.H. Nguyen, B.-J. Lee, Protein corona: a new approach for nanomedicine design, Int. J. Nanomed. 12 (2017) 3137 3151. Available from: https://doi.org/10.2147/IJN.S129300. PubMed PMID: PMC5402904. [13] S. Nie, Understanding and overcoming major barriers in cancer nanomedicine, Nanomedicine 5 (4) (2010) 523 528. Available from: https://doi.org/10.2217/nnm.10.23. PubMed PMID: PMC2913868. [14] M.M. Frank, L.F. Fries, The role of complement in inflammation and phagocytosis, Immunol. Today 12 (9) (1991) 322 326. Available from: https://doi.org/10.1016/0167-5699(91)90009-I. [15] D.E. Owens, N.A. Peppas, Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles, Int. J. Pharm. 307 (1) (2006) 93 102. Available from: https://doi.org/10.1016/j.ijpharm.2005.10.010. [16] J.S. Suk, Q. Xu, N. Kim, J. Hanes, L.M. Ensign, PEGylation as a strategy for improving nanoparticlebased drug and gene delivery, Adv. Drug Deliv. Rev. 99 (Pt A) (2016) 28 51. Available from: https:// doi.org/10.1016/j.addr.2015.09.012. PubMed PMID: 26456916; PMCID: PMC4798869. [17] A.J. Cole, V.C. Yang, A.E. David, Cancer theranostics: the rise of targeted magnetic nanoparticles, Trends Biotechnol. 29 (7) (2011) 323 332. Available from: https://doi.org/10.1016/j.tibtech.2011.03.001. PubMed PMID: PMC3210200. [18] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review, J. Control. Release 65 (1-2) (2000) 271 284. PubMed PMID: 10699287. [19] B. Romberg, W.E. Hennink, G. Storm, Sheddable coatings for long-circulating nanoparticles, Pharm. Res. 25 (1) (2008) 55 71. Available from: https://doi.org/10.1007/s11095-007-9348-7. PubMed PMID: 17551809; PMCID: PMC2190344. [20] T. Ishida, M. Ichihara, X. Wang, K. Yamamoto, J. Kimura, E. Majima, et al., Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes, J. Control. Release 112 (1) (2006) 15 25. Available from: https://doi.org/10.1016/ j.jconrel.2006.01.005. [21] Z. Amoozgar, Y. Yeo, Recent advances in stealth coating of nanoparticle drug delivery systems, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 4 (2) (2012) 219 233. Available from: https://doi.org/ 10.1002/wnan.1157. PubMed PMID: 22231928; PMCID: PMC3288878. [22] W. Yang, J.-R. Ella-Menye, T. Bai, A. Sinclair, S. Jiang, Stable and functionalizable quantum dots with a thin zwitterionic carboxybetaine layer, Langmuir 33 (35) (2017) 8784 8789. Available from: https://doi. org/10.1021/acs.langmuir.7b00981.

Chapter 2 • Biodistribution, Excretion, and Toxicity of Nanoparticles

49

[23] J. An, Q. Guo, P. Zhang, A. Sinclair, Y. Zhao, X. Zhang, et al., Hierarchical design of a polymeric nanovehicle for efficient tumor regression and imaging, Nanoscale 8 (17) (2016) 9318 9327. Available from: https://doi.org/10.1039/C6NR01595F. [24] C.-J. Huang, S.-H. Chu, L.-C. Wang, C.-H. Li, T.R. Lee, Bioinspired zwitterionic surface coatings with robust photostability and fouling resistance, ACS Appl. Mater. Interfaces 7 (42) (2015) 23776 23786. Available from: https://doi.org/10.1021/acsami.5b08418. [25] S. Muro, C. Garnacho, J.A. Champion, J. Leferovich, C. Gajewski, E.H. Schuchman, et al., Control of endothelial targeting and intracellular delivery of therapeutic enzymes by modulating the size and shape of ICAM-1-targeted carriers, Mol. Ther. 16 (8) (2008) 1450 1458. Available from: https://doi.org/ 10.1038/mt.2008.127. PubMed PMID: 18560419; PMCID: PMC2810502. [26] L.L. Rouhana, J.A. Jaber, J.B. Schlenoff, Aggregation-resistant water-soluble gold nanoparticles, Langmuir 23 (26) (2007) 12799 12801. Available from: https://doi.org/10.1021/la702151q. [27] S. Sun, Y. Huang, C. Zhou, S. Chen, M. Yu, J. Liu, et al., Effect of hydrophobicity on nano-bio interactions of zwitterionic luminescent gold nanoparticles at the cellular level, Bioconjugate Chem. 29 (6) (2018) 1841 1846. Available from: https://doi.org/10.1021/acs.bioconjchem.8b00202. PubMed PMID: 29775044. [28] T. Cedervall, I. Lynch, S. Lindman, T. Berggård, E. Thulin, H. Nilsson, et al., Understanding the nanoparticle protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles, Proc. Natl. Acad. Sci. U.S.A. 104 (7) (2007) 2050. [29] P.R. Leroueil, S.A. Berry, K. Duthie, G. Han, V.M. Rotello, D.Q. McNerny, et al., Wide varieties of cationic nanoparticles induce defects in supported lipid bilayers, Nano Lett. 2 (8) (2008) 420 424. Available from: https://doi.org/10.1021/nl0722929. PubMed PMID: 18217783. [30] E.C. Cho, J. Xie, P.A. Wurm, Y. Xia, Understanding the role of surface charges in cellular adsorption versus internalization by selectively removing gold nanoparticles on the cell surface with a I2/KI etchant, Nano Lett. 9 (3) (2009) 1080 1084. Available from: https://doi.org/10.1021/nl803487r. PubMed PMID: 19199477. [31] T.S. Levchenko, R. Rammohan, A.N. Lukyanov, K.R. Whiteman, V.P. Torchilin, Liposome clearance in mice: the effect of a separate and combined presence of surface charge and polymer coating, Int. J. Pharm. 240 (1) (2002) 95 102. Available from: https://doi.org/10.1016/S0378-5173(02)00129-1. [32] C. He, Y. Hu, L. Yin, C. Tang, C. Yin, Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles, Biomaterials 31 (13) (2010) 3657 3666. Available from: https://doi.org/10.1016/j.biomaterials.2010.01.065. [33] J. Jin, Y.-J. Gu, C.W.-Y. Man, J. Cheng, Z. Xu, Y. Zhang, et al., Polymer-coated NaYF4:Yb3 1 , Er3 1 upconversion nanoparticles for charge-dependent cellular imaging, ACS Nano 5 (10) (2011) 7838 7847. Available from: https://doi.org/10.1021/nn201896m. [34] D. Sultan, D. Ye, G.S. Heo, X. Zhang, H. Luehmann, Y. Yue, et al., Focused ultrasound enabled trans-blood brain barrier delivery of gold nanoclusters: effect of surface charges and quantification using positron emission tomography, Small 14 (30) (2018) e1703115. Available from: https://doi.org/ 10.1002/smll.201703115. PubMed PMID: 29966035. [35] H.-S. Han, J.D. Martin, J. Lee, D.K. Harris, D. Fukumura, R.K. Jain, et al., Spatial charge configuration regulates nanoparticle transport and binding behavior in vivo, Angew. Chem. Int. Ed. 52 (5) (2013) 1414 1419. Available from: https://doi.org/10.1002/anie.201208331. [36] A.K. Murthy, R.J. Stover, W.G. Hardin, R. Schramm, G.D. Nie, S. Gourisankar, et al., Charged gold nanoparticles with essentially zero serum protein adsorption in undiluted fetal bovine serum, J. Am. Chem. Soc. 135 (21) (2013) 7799 7802. Available from: https://doi.org/10.1021/ja400701c. [37] S. Hak, E. Helgesen, H.H. Hektoen, E.M. Huuse, P.A. Jarzyna, W.J.M. Mulder, et al., The effect of nanoparticle polyethylene glycol surface density on ligand-directed tumor targeting studied in vivo by dual modality imaging, ACS Nano 6 (6) (2012) 5648 5658. Available from: https://doi.org/10.1021/ nn301630n.

50

THERANOSTIC BIONANOMATERIALS

[38] S.G. Elci, Y. Jiang, B. Yan, S.T. Kim, K. Saha, D.F. Moyano, et al., Surface charge controls the suborgan biodistributions of gold nanoparticles, ACS Nano 10 (5) (2016) 5536 5542. Available from: https://doi. org/10.1021/acsnano.6b02086. [39] S.M. Moghimi, H. Hedeman, I.S. Muir, L. Illum, S.S. Davis, An investigation of the filtration capacity and the fate of large filtered sterically-stabilized microspheres in rat spleen, Biochim. Biophys. Acta 1157 (3) (1993) 233 240. PubMed PMID: 8323953. [40] H.S. Choi, W. Liu, P. Misra, E. Tanaka, J.P. Zimmer, B. Itty Ipe, et al., Renal clearance of quantum dotsNat. Biotechnol. 25 (10) (2007) 1165 1170. Available from: https://doi.org/10.1038/nbt1340PubMed PMID: 17891134; PMCID: PMC2702539 . Available from: https://www.nature.com/articles/nbt1340#supplementary-information. [41] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Res. 46 (12 Part1) (1986) 6387 6392. [42] S. Wilhelm, A.J. Tavares, Q. Dai, S. Ohta, J. Audet, H.F. Dvorak, et al., Analysis of nanoparticle delivery to tumours, Nat. Rev. Mater. 1 (2016) 16014. Available from: https://doi.org/10.1038/natrevmats.2016.14. Available from: https://www.nature.com/articles/natrevmats201614#supplementary-information. [43] G.M. Soliman, A. Sharma, D. Maysinger, A. Kakkar, Dendrimers and miktoarm polymers based multivalent nanocarriers for efficient and targeted drug delivery, Chem. Commun. 47 (34) (2011) 9572 9587. Available from: https://doi.org/10.1039/C1CC11981H. [44] Y. Wang, Y. Liu, H. Luehmann, X. Xia, P. Brown, C. Jarreau, et al., Evaluating the pharmacokinetics and in vivo cancer targeting capability of Au nanocages by positron emission tomography imaging, ACS Nano 6 (7) (2012) 5880 5888. Available from: https://doi.org/10.1021/nn300464r. [45] D. Liu, A. Mori, L. Huang, Role of liposome size and RES blockade in controlling biodistribution and tumor uptake of GM1-containing liposomes, Biochim. Biophys. Acta 1104 (1) (1992) 95 101. Available from: https://doi.org/10.1016/0005-2736(92)90136-A. [46] Y. Zhao, D. Sultan, L. Detering, S. Cho, G. Sun, R. Pierce, et al., Copper-64-alloyed gold nanoparticles for cancer imaging: improved radiolabel stability and diagnostic accuracy, Angew. Chem. Int. Ed. 53 (1) (2014) 156 159. Available from: https://doi.org/10.1002/anie.201308494. [47] Y. Zhao, D. Sultan, L. Detering, H. Luehmann, Y. Liu, Facile synthesis, pharmacokinetic and systemic clearance evaluation, and positron emission tomography cancer imaging of 64Cu-Au alloy nanoclusters, Nanoscale 6 (22) (2014) 13501 13509. Available from: https://doi.org/10.1039/C4NR04569F. [48] N. Hoshyar, S. Gray, H. Han, G. Bao, The effect of nanoparticle size on in vivo pharmacokinetics and cellular interaction, Nanomedicine 11 (6) (2016) 673 692. Available from: https://doi.org/10.2217/nnm.16.5. [49] Y. Wang, K.C.L. Black, H. Luehmann, W. Li, Y. Zhang, X. Cai, et al., Comparison study of gold nanohexapods, nanorods, and nanocages for photothermal cancer treatment, ACS Nano 7 (3) (2013) 2068 2077. Available from: https://doi.org/10.1021/nn304332s. [50] K.C.L. Black, Y. Wang, H.P. Luehmann, X. Cai, W. Xing, B. Pang, et al., Radioactive 198Au-doped nanostructures with different shapes for in vivo analyses of their biodistribution, tumor uptake, and intratumoral distribution, ACS Nano 8 (5) (2014) 4385 4394. Available from: https://doi.org/10.1021/nn406258m. [51] S. Shukla, F.J. Eber, A.S. Nagarajan, N.A. DiFranco, N. Schmidt, A.M. Wen, et al., The impact of aspect ratio on the biodistribution and tumor homing of rigid soft-matter nanorods, Adv. Healthcare Mater. 4 (6) (2015) 874 882. Available from: https://doi.org/10.1002/adhm.201400641. [52] D.L. Jasinski, H. Li, P. Guo, The effect of size and shape of RNA nanoparticles on biodistribution, Mol. Ther. 26 (3) (2018) 784 792. Available from: https://doi.org/10.1016/j.ymthe.2017.12.018. [53] S. Takeoka, Y. Teramura, Y. Okamura, E. Tsuchida, M. Handa, Y. Ikeda, Rolling properties of rGPIbαconjugated phospholipid vesicles with different membrane flexibilities on vWf surface under flow conditions, Biochem. Biophys. Res. Commun. 296 (3) (2002) 765 770. Available from: https://doi.org/ 10.1016/S0006-291X(02)00934-8.

Chapter 2 • Biodistribution, Excretion, and Toxicity of Nanoparticles

51

[54] L. Zhang, Z. Cao, Y. Li, J.-R. Ella-Menye, T. Bai, S. Jiang, Softer zwitterionic nanogels for longer circulation and lower splenic accumulation, ACS Nano 6 (8) (2012) 6681 6686. Available from: https:// doi.org/10.1021/nn301159a. [55] X. Sun, R. Rossin, J.L. Turner, M.L. Becker, M.J. Joralemon, M.J. Welch, et al., An assessment of the effects of shell cross-linked nanoparticle size, core composition, and surface PEGylation on in vivo biodistribution, Biomacromolecules 6 (5) (2005) 2541 2554. Available from: https://doi.org/10.1021/bm050260e. [56] J. Yu, W. Yin, T. Peng, Y.-N. Chang, Y. Zu, J. Li, et al., Biodistribution, excretion, and toxicity of polyethyleneimine modified NaYF4:Yb, Er upconversion nanoparticles in mice via different administration routes, Nanoscale 9 (13) (2017) 4497 4507. Available from: https://doi.org/10.1039/C7NR00078B. [57] X.-D. Zhang, H.-Y. Wu, D. Wu, Y.-Y. Wang, J.-H. Chang, Z.-B. Zhai, et al., Toxicologic effects of gold nanoparticles in vivo by different administration routes, Int. J. Nanomed. 5 (2010) 771 781. Available from: https://doi.org/10.2147/IJN.S8428. PubMed PMID: PMC2962273. [58] H. Xie, B. Goins, A. Bao, Z.J. Wang, W.T. Phillips, Effect of intratumoral administration on biodistribution of (64)Cu-labeled nanoshells, Int. J. Nanomed. 7 (2012) 2227 2238. Available from: https://doi.org/ 10.2147/IJN.S30699. PubMed PMID: PMC3356223. [59] X. Huang, F. Zhang, L. Zhu, K.Y. Choi, N. Guo, J. Guo, et al., The effect of injection routes on the biodistribution, clearance and tumor uptake of carbon dots, ACS Nano 7 (7) (2013) 5684 5693. Available from: https://doi.org/10.1021/nn401911k. PubMed PMID: PMC3725601. [60] S.-Y. Lee, J.-X. Cheng, Clearance of Nanoparticles During Circulation. Pharmaceutical Sciences Encyclopedia: Nanoparticulate Drug Delivery Systems: Strategies, Technologies, and Applications, John Wiley & Sons, Inc., 2014, pp. 209 240. Available from: http://dx.doi.org/10.1002/9780470571224.pse496. [61] T. Bose, D. Latawiec, P.P. Mondal, S. Mandal, Overview of nano-drugs characteristics for clinical application: the journey from the entry to the exit point, J. Nanopart. Res. 16 (8) (2014) 2527. Available from: https://doi.org/10.1007/s11051-014-2527-7. [62] K.M. Tsoi, S.A. MacParland, X.-Z. Ma, V.N. Spetzler, J. Echeverri, B. Ouyang, et al., Mechanism of hardnanomaterial clearance by the liver, Nat. Mater. 15 (2016) 1212. Available from: https://doi.org/10.1038/ nmat4718. Available from: https://www.nature.com/articles/nmat4718#supplementary-information. [63] J.V. Jokerst, T. Lobovkina, R.N. Zare, S.S. Gambhir, Nanoparticle PEGylation for imaging and therapy, Nanomedicine 6 (4) (2011) 715 728. Available from: https://doi.org/10.2217/nnm.11.19. PubMed PMID: 21718180. [64] M. Yu, J. Zheng, Clearance pathways and tumor targeting of imaging nanoparticles, ACS Nano 9 (7) (2015) 6655 6674. Available from: https://doi.org/10.1021/acsnano.5b01320. [65] N. Khlebtsov, L. Dykman, Biodistribution and toxicity of engineered gold nanoparticles: a review of in vitro and in vivo studies, Chem. Soc. Rev. 40 (3) (2011) 1647 1671. Available from: https://doi.org/ 10.1039/C0CS00018C. [66] K.-T. Yong, W.-C. Law, R. Hu, L. Ye, L. Liu, M.T. Swihart, et al., Nanotoxicity assessment of quantum dots: from cellular to primate studies, Chem. Soc. Rev. 42 (3) (2013) 1236 1250. Available from: https:// doi.org/10.1039/C2CS35392J. [67] E. Sadauskas, G. Danscher, M. Stoltenberg, U. Vogel, A. Larsen, H. Wallin, Protracted elimination of gold nanoparticles from mouse liver, Nanomed. Nanotechnol. Biol. Med. 5 (2) (2009) 162 169. Available from: https://doi.org/10.1016/j.nano.2008.11.002. [68] L. Ye, K.-T. Yong, L. Liu, I. Roy, R. Hu, J. Zhu, et al., A pilot study in non-human primates shows no adverse response to intravenous injection of quantum dots, Nat. Nanotechnol. 7 (2012) 453. Available from: https://doi.org/10.1038/nnano.2012.74. Available from: https://www.nature.com/articles/nnano.2012.74#supplementary-information. [69] B. Ballou, L.A. Ernst, S. Andreko, T. Harper, J.A.J. Fitzpatrick, A.S. Waggoner, et al., Sentinel lymph node imaging using quantum dots in mouse tumor models, Bioconjugate Chem. 18 (2) (2007) 389 396. Available from: https://doi.org/10.1021/bc060261j.

52

THERANOSTIC BIONANOMATERIALS

[70] R. Kumar, I. Roy, T.Y. Ohulchanskky, L.A. Vathy, E.J. Bergey, M. Sajjad, et al., In vivo biodistribution and clearance studies using multimodal organically modified silica nanoparticles, ACS Nano 4 (2) (2010) 699 708. Available from: https://doi.org/10.1021/nn901146y. [71] L. Gu, R.H. Fang, M.J. Sailor, J.-H. Park, In vivo clearance and toxicity of monodisperse iron oxide nanocrystals, ACS Nano 6 (6) (2012) 4947 4954. Available from: https://doi.org/10.1021/nn300456z. [72] J.P.M. Almeida, A.L. Chen, A. Foster, R. Drezek, In vivo biodistribution of nanoparticles, Nanomedicine 6 (5) (2011) 815 835. Available from: https://doi.org/10.2217/nnm.11.79. PubMed PMID: 21793674. [73] W. Liu, H.S. Choi, J.P. Zimmer, E. Tanaka, J.V. Frangioni, M. Bawendi, Compact cysteine-coated CdSe (ZnCdS) quantum dots for in vivo applications, J. Am. Chem. Soc. 129 (47) (2007) 14530 14531. Available from: https://doi.org/10.1021/ja073790m. [74] H.S. Choi, B.I. Ipe, P. Misra, J.H. Lee, M.G. Bawendi, J.V. Frangioni, Tissue- and organ-selective biodistribution of NIR fluorescent quantum dots, Nano Lett. 9 (6) (2009) 2354 2359. Available from: https:// doi.org/10.1021/nl900872r. [75] Y. Zhao, L. Detering, D. Sultan, M.L. Cooper, M. You, S. Cho, et al., Gold nanoclusters doped with 64Cu for CXCR4 positron emission tomography imaging of breast cancer and metastasis, ACS Nano 10 (6) (2016) 5959 5970. Available from: https://doi.org/10.1021/acsnano.6b01326. [76] C. Fu, T. Liu, L. Li, H. Liu, D. Chen, F. Tang, The absorption, distribution, excretion and toxicity of mesoporous silica nanoparticles in mice following different exposure routes, Biomaterials 34 (10) (2013) 2565 2575. Available from: https://doi.org/10.1016/j.biomaterials.2012.12.043. [77] X. He, H. Nie, K. Wang, W. Tan, X. Wu, P. Zhang, In vivo study of biodistribution and urinary excretion of surface-modified silica nanoparticles, Anal. Chem. 80 (24) (2008) 9597 9603. Available from: https:// doi.org/10.1021/ac801882g. [78] X. Huang, L. Li, T. Liu, N. Hao, H. Liu, D. Chen, et al., The shape effect of mesoporous silica nanoparticles on biodistribution, clearance, and biocompatibility in vivo, ACS Nano 5 (7) (2011) 5390 5399. Available from: https://doi.org/10.1021/nn200365a. [79] M. Gary-Bobo, Y. Mir, C. Rouxel, D. Brevet, I. Basile, M. Maynadier, et al., Mannose-functionalized mesoporous silica nanoparticles for efficient two-photon photodynamic therapy of solid tumors, Angew. Chem. Int. Ed. 50 (48) (2011) 11425 11429. Available from: https://doi.org/10.1002/anie.201104765. [80] A.L. Ginzburg, L. Truong, R.L. Tanguay, J.E. Hutchison, Synergistic toxicity produced by mixtures of biocompatible gold nanoparticles and widely used surfactants, ACS Nano 12 (6) (2018) 5312 5322. Available from: https://doi.org/10.1021/acsnano.8b00036. [81] Y.-S. Chen, Y.-C. Hung, I. Liau, G.S. Huang, Assessment of the in vivo toxicity of gold nanoparticles, Nanoscale Res. Lett. 4 (8) (2009) 858 864. Available from: https://doi.org/10.1007/s11671-009-9334-6. [82] W.-S. Cho, M. Cho, J. Jeong, M. Choi, H.-Y. Cho, B.S. Han, et al., Acute toxicity and pharmacokinetics of 13 nm-sized PEG-coated gold nanoparticles, Toxicol. Appl. Pharmacol. 236 (1) (2009) 16 24. Available from: https://doi.org/10.1016/j.taap.2008.12.023. [83] X.-D. Zhang, D. Wu, X. Shen, P.-X. Liu, N. Yang, B. Zhao, et al., Size-dependent in vivo toxicity of PEG-coated gold nanoparticles, Int. J. Nanomed. 6 (2011) 2071 2081. Available from: https://doi.org/ 10.2147/IJN.S21657. PubMed PMID: PMC3181066. [84] G. Liu, J. Gao, H. Ai, X. Chen, Applications and potential toxicity of magnetic iron oxide nanoparticles, Small 9 (9 10) (2013) 1533 1545. Available from: https://doi.org/10.1002/smll.201201531. [85] Y. Wei, M. Zhao, F. Yang, Y. Mao, H. Xie, Q. Zhou, Iron overload by superparamagnetic iron oxide nanoparticles is a high risk factor in cirrhosis by a systems toxicology assessment, Sci. Rep. 6 (2016) 29110. Available from: https://doi.org/10.1038/srep29110. Available from: https://www.nature.com/articles/srep29110#supplementary-information. [86] A.M. Derfus, W.C.W. Chan, S.N. Bhatia, Probing the cytotoxicity of semiconductor quantum dots, Nano Lett. 4 (1) (2004) 11 18. Available from: https://doi.org/10.1021/nl0347334.

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[87] M.J.D. Clift, J. Varet, S.M. Hankin, B. Brownlee, A.M. Davidson, C. Brandenberger, et al., Quantum dot cytotoxicity in vitro: an investigation into the cytotoxic effects of a series of different surface chemistries and their core/shell materials, Nanotoxicology 5 (4) (2011) 664 674. Available from: https://doi.org/ 10.3109/17435390.2010.534196. [88] I. Iavicoli, L. Fontana, G. Nordberg, The effects of nanoparticles on the renal system, Crit. Rev. Toxicol. 46 (6) (2016) 490 560. Available from: https://doi.org/10.1080/10408444.2016.1181047. [89] K. Rana, Y. Verma, V. Rani, S.V.S. Rana, Renal toxicity of nanoparticles of cadmium sulphide in rat, Chemosphere 193 (2018) 142 150. Available from: https://doi.org/10.1016/j.chemosphere.2017.11.011. [90] Q. Feng, Y. Liu, J. Huang, K. Chen, J. Huang, K. Xiao, Uptake, distribution, clearance, and toxicity of iron oxide nanoparticles with different sizes and coatings, Sci. Rep. 8 (1) (2018) 2082. Available from: https://doi.org/10.1038/s41598-018-19628-z. [91] H.S. Choi, W. Liu, P. Misra, E. Tanaka, J.P. Zimmer, B. Itty Ipe, et al., Renal clearance of quantum dots, Nat. Biotechnol. 25 (2007) 1165 1170. Available from: https://doi.org/10.1038/nbt1340. [92] B. Du, X. Jiang, A. Das, Q. Zhou, M. Yu, R. Jin, et al., Glomerular barrier behaves as an atomically precise bandpass filter in a sub-nanometre regime, Nat. Nanotechnol. 12 (2017) 1096. Available from: https://doi.org/10.1038/nnano.2017.170. Available from: https://www.nature.com/articles/ nnano.2017.170#supplementary-information. [93] C. Yang, A. Tian, Z. Li, Reversible cardiac hypertrophy induced by PEG-coated gold nanoparticles in mice, Sci. Rep. 6 (2016) 20203. Available from: https://doi.org/10.1038/srep20203. Available from: https://www.nature.com/articles/srep20203#supplementary-information. [94] M.A. Abdelhalim, Exposure to gold nanoparticles produces cardiac tissue damage that depends on the size and duration of exposure, Lipids Health Dis. 10 (2011) 205. Available from: https://doi.org/ 10.1186/1476-511x-10-205. PubMed PMID: 22073987; PMCID: PMC3278471. [95] C.-X. Lin, S.-Y. Yang, J.-L. Gu, J. Meng, H.-Y. Xu, J.-M. Cao, The acute toxic effects of silver nanoparticles on myocardial transmembrane potential, INa and IK1 channels and heart rhythm in mice, Nanotoxicology 11 (6) (2017) 827 837. Available from: https://doi.org/10.1080/17435390.2017.1367047. [96] D. Ye, D. Sultan, X. Zhang, Y. Yue, G.S. Heo, S. Kothapalli, et al., Focused ultrasound-enabled delivery of radiolabeled nanoclusters to the pons, J. Control. Release 283 (2018) 143 150. Available from: https://doi.org/10.1016/j.jconrel.2018.05.039. PubMed PMID: 29864474; PMCID: PMC6035767. [97] Y. Zhou, Z. Peng, E.S. Seven, R.M. Leblanc, Crossing the blood-brain barrier with nanoparticles, J. Control. Release 270 (2018) 290 303. Available from: https://doi.org/10.1016/j.jconrel.2017.12.015. PubMed PMID: 29269142. [98] G. Oberdorster, A. Elder, A. Rinderknecht, Nanoparticles and the brain: cause for concern? J. Nanosci. Nanotechnol. 9 (8) (2009) 4996 5007. PubMed PMID: 19928180; PMCID: PMC3804071. [99] C. Carlson, S.M. Hussain, A.M. Schrand, L.K. Braydich-Stolle, K.L. Hess, R.L. Jones, et al., Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species, J. Phys. Chem. B 112 (43) (2008) 13608 13619. Available from: https://doi.org/10.1021/jp712087m. PubMed PMID: 18831567. [100] S. Lanone, F. Rogerieux, J. Geys, A. Dupont, E. Maillot-Marechal, J. Boczkowski, et al., Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines, Part. Fibre Toxicol. 6 (2009) 14. Available from: https://doi.org/10.1186/1743-8977-6-14. PubMed PMID: 19405955; PMCID: PMC2685765. [101] X. Lai, H. Zhao, Y. Zhang, K. Guo, Y. Xu, S. Chen, et al., Intranasal delivery of copper oxide nanoparticles induces pulmonary toxicity and fibrosis in C57BL/6 mice, Sci. Rep. 8 (1) (2018) 4499. Available from: https://doi.org/10.1038/s41598-018-22556-7. PubMed PMID: 29540716; PMCID: PMC5852054. [102] M.J. Ernsting, M. Murakami, A. Roy, S.-D. Li, Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles, J. Control. Release 172 (3) (2013) 782 794. Available from: https://doi.org/10.1016/j.jconrel.2013.09.013.

3 Nanoparticle Interaction With Immune Cells for NanoparticleMediated (Anticancer) Immunotherapy Per Hydbring1, Juan Du2 1

D E P AR T MEN T O F O NC O L O GY AN D PA T HO L OGY, VISIONSGATAN 4 , K AROLINSKA INS TIT UT ET , S -17 16 4 ST OCK HOLM , SW EDEN 2 DEPARTMENT OF MICROBIOLOGY, TUMOR AND CELL BIOLOGY, CENT RE FOR TRANSLATIONAL MICROBIOME R ESEARCH (CTMR ), VISIONSGATAN 4, KAROLINSKA I NSTITUT ET , S -17 16 4 STO CK HOLM, SW EDEN

3.1 Introduction 3.1.1 Nanoparticles Nanoparticles are vesicular-like vehicles with a diameter of less than 1 μm. Their properties include systemic stability, target site specificity, and level of solubility. These properties are mainly affected by the molecular structure of the vehicle surface along with the size and shape of the particle, which can all be modified through customized synthetic chemistry [1,2], providing a vast number of possible downstream biological approaches [1,2]. The field of nanoparticles has been thoroughly summarized in numerous review articles [3,4]. However, nanoparticles have not until recently been extensively exploited for their ability to tune the response output of immune cells. Before going deeper into the various examples of immune cell targeting by nanoparticles, it is important to briefly introduce the different classes of nanoparticles. While many nanoparticles used for therapeutic applications are synthetic, there are also naturally formed nanoparticles used for such purposes. The main classes of therapeutic nanoparticles are: liposomal nanoparticles, dendritic nanoparticles, metal-based nanoparticles, silica-based nanoparticles, and carbon-based nanoparticles (Fig. 3 1). The surface properties (e.g., charge and solubility) along with the particle size determine target specificity and efficiency (Fig. 3 2). Liposomal nanoparticles are popular for the delivery of nucleic acid material since their properties protect the negatively charged DNA while allowing for cellular uptake. The major drawback with liposomal nanoparticles is their limited systemic stability. Hence, a liposomal nanoparticle may be an excellent choice for a particular cell type but a poor choice for another. Dendritic nanoparticles are readily Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00003-1 © 2019 Elsevier Inc. All rights reserved.

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FIGURE 3–1 Schematic representation of the main classes of nanoparticles [5]. Reprinted/adapted by permission from the Royal Society of Chemistry: Elsevier. Theranostic Bionanomaterials by Juan Du and Per Hydbring. Elsevier Ltd. All rights reserved. 2018.

FIGURE 3–2 Nanoparticle properties and impact on the immune system [6]. Reprinted/adapted by permission from Springer Nature: Elsevier. Theranostic Bionanomaterials by Juan Du and Per Hydbring. Elsevier Ltd. All rights reserved. 2018.

used for encapsulation of drugs due to basic preparation protocols. However, dendritic nanoparticles generally possess low solubility in water with concomitant toxicity, requiring customized modifications. Another popular nanoparticle class is metal-based nanoparticles. Synthesis of metal-based nanoparticles is straightforward and easily customized. However, as with dendritic nanoparticles, toxicity is a major concern, although for distinct reasons. Metal-based nanoparticles may penetrate the cell nucleus and have been associated with reports of increased oxidative stress. Silica-based nanoparticles can be used for a large

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variety of cargo encapsulations. They are overall considered to possess low toxicity and high stability, but as with most nanoparticle classes, toxicity is highly influenced by particle size and surface charge. Carbon-based nanoparticles are hydrophobic particles that cannot be employed unmodified due to insolubility. Following surface modification they can be used for carrying a variety of cargoes. Overall, the degree of surface modification will dictate the stability and biodistribution of carbon-based nanoparticles, making it difficult to compare them in generalized terms to other classes of nanoparticles. However, if properly functionalized they are considered to possess high delivery efficacy with limited toxicity [6]. The biological repertoire of nanoparticles used to target the immune system includes not only the transportation of modulating agents but also vaccination therapies. Further, while this chapter focuses on cancer, there are various other diseases investigated for immunomodulatory nanoparticles [7 11]. A range of physical properties, including elasticity, density, shape, charge, size, and surface functionalization of nanoparticles dictate their efficacy for cellular internalization, vascular transportation as well as impact on the immune system, and they have been extensively described elsewhere [12]. In this chapter, we focus on the biological output resulting from the interaction between nanoparticles and the immune system.

3.1.2 Innate Immune System What is the best biological approach to target immune cells for anticancer therapy? This depends on the particular cancer disease, the genetic/epigenetic landscape of immune system components in that cancer, and whether the cancer has developed any addiction to that landscape signature. Further, it is unlikely that modulation of any immune system component is equally efficient for a particular cancer. Therefore, in order to tailor a nanoparticle immune system therapy, we first have to understand the biological basis of the immune system. Using a very crude categorization, the immune system consists of innate immunity and adaptive immunity. While the innate immunity responds very rapidly to intruding molecules, its response is rather nonspecific. It commences when innate immune cells utilize, among other receptors, pattern recognition receptors (PRRs) to bind pathogenassociated molecular pattern (PAMP) molecules [13,14], initiating a signaling cascade leading to substantial gene expression alterations with the output of altered secretion of chemokines and cytokines. Such secretion attracts specific immune cells belonging to the innate immune system, including neutrophils, macrophages, and natural killer cells, leading to elimination of pathogens [13,14]. In contrast, adaptive immunity executes a slower but more focused response initiating from the innate immunity or from specific exhibition of antigens. The exhibition of antigens is facilitated by so-called major histocompatibility complex (MHC) molecules. MHC molecules are presented on macrophages and dendritic cells (DCs) but not on other immune cells. A common name for cells harboring the ability to present antigens is antigen-presenting cells (APCs) [15 17]. Although nanoparticle therapy can be tailored in a way that specific immune components are targeted, it is crucial to remember that a change in an individual immune component may completely alter immune system pathway signaling since components of the innate and adaptive immunity are closely connected.

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For example, in addition to their role as APCs, DCs express receptors of importance for innate immunity as well. Therefore, they possess the biological properties to connect innate immunity with adaptive immunity [15 17]. In order to trigger a response of the innate immune system, nanoparticles are often engulfed by APCs. Through their PRRs [e.g., C-type lectin receptors (CLRs), Toll-like receptors (TLRs), RIG-I-like receptors, NOD-like receptors (NLRs)], macrophages and DCs interact with a variety of PAMPs [15 17]. Nanoparticle targeting of APCs usually involves specific targeting of TLRs, although a limited number of studies have investigated targeting of NLRs and CLRs as well. As for TLRs, nanoparticles may interact with both intracellular receptors and surface receptors (Fig. 3 3). No matter which specific TLR, the output will be a significant immune system boost, resulting in activation of the innate immune system [19 21].

FIGURE 3–3 Schematics of nanoparticle uptake by APCs, and induction of innate immunity. APCs including DCs express PRRs, for example, TLR, NLRs, RLRs, and CLRs, which are localized either at the plasma membrane or intracellularly. PRRs, which recognize PAMP molecules, can be targeted by nanoparticles. The activation of PRRs recruits adaptors and ligands to trigger downstream signaling. This results in release of inflammatory cytokines and activation of additional cells belonging to innate immunity [18]. APCs, Antigen-presenting cells; CLRs, C-type lectin receptors; DCs, dendritic cells; NLRs, NOD-like receptors; PAMP, pathogen-associated molecular pattern; PRRs, pattern recognition receptors; RLRs, RIG-I-like receptors; TLR, Toll-like receptors. Reprinted/adapted by permission from Elservier: Elsevier. Theranostic Bionanomaterials by Juan Du and Per Hydbring. Elsevier Ltd. All rights reserved. 2018.

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3.1.3 Adaptive Immune System As previously mentioned, the adaptive immune system executes slower but more specific responses to foreign substances. The adaptive immune system consists of specialized lymphocytes called B and T cells. Activated B cells, so called plasma cells, produce highly specific antibodies binding to foreign antigens. Activated T cells recognize these antigens after engulfment by APCs and exhibition through MHCs. There are numerous subgroups of T cells with the most dominant being CD8 1 T cells and CD4 1 T cells executing distinct responses to external stimuli, partly due to their activation of the T cell receptor (TCR) by different types of MHCs. CD8 1 T cells are activated by MHC-I molecules while CD4 1 T cells are activated by MHC-II molecules. For a successful nanoparticle therapeutic approach, it is necessary to understand the nature of these adaptive immune cells [15 17]. As mentioned above, activated B cells are the main producers of antibodies, which in turn bind antigens and are taken up by APCs. Antibodies, or immunoglobulins (Ig), are divided into five major classes; IgA, D, E, G, M, each possessing an enormously extensive antigen recognition repertoire [15 17]. Through this antibody library, B cells communicate with T cells. CD8 1 T cells, also referred to as cytotoxic T cells, are activated through direct antigen presentation by unhealthy cells, including cancer cells [15 17]. For the utilization of nanoparticles in anticancer therapeutics, many investigations involve modulation of the responses of the T cells [22], T cells migrate to the site of the tumor [23], specific activation of T cells [24], as well as help nanoparticles alter the proportion of T cell subtypes [25,26]. The antitumor immune response of CD4 1 T cells is greatly influenced by their cytokines and the activation of CD4 1 T cell membrane receptors [27]. As shown in Fig. 3 4, naïve T cells can be differentiated into large subsets of CD4 1 T cells. T helper 1 (Th1) and T helper 2 (Th2) cells produce IFN-γ and interleukin (IL)-4, which keep the balance in the microenvironment of cellular and humoral immunity responses for the clearance of cancer cells [28]. Regulatory T cells (Treg), on the other hand, are considered as powerful inhibitors of antitumor immunity response by IL-10 and TGF-β1 secretion. These cytokines block the activation of DC cells and the antitumor responses from effector T cells such as Th1 and Th2 [29,30]. Strategies in immunotherapy have been investigated for modulation of Treg cells [31]. Again, modulation of the adaptive immune system by nanoparticles may either occur through direct nanoparticle uptake to a specialized cell type, or through a nonspecific APC uptake. In order for an effect to be limited to a specific cell type, the nanoparticle targeting would need to happen further down the immune system cascade, for example, to activated T cells, and would require protein adaptors highly specific for that cell type conjugated on the nanoparticle shell [15 17]. In summary, the immune system can be targeted by nanoparticles either in a nonspecific or highly specific way. The end output is that all approaches somehow enhance the responses of activated T cells. The chemistry of the nanoparticle is essential for the level of specificity and efficacy in any immune system targeting approach.

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FIGURE 3–4 Schematics of nanoparticle-mediated activation of adaptive immunity. Nanoparticles activate the maturation of T cells and B cells either indirectly, through uptake by APCs, or directly, by targeting surface receptors on T cells and B cells. Activation of adaptive immune cells requires the antigens to be presented through MHC molecules. After maturation, both T cells and B cells differentiate into multiple subpopulations, including memory cells, which are essential for long-term immune responses. In addition, B cells differentiate into plasma cells, which generate antigen-specific antibodies used to neutralize foreign substances. Differentiation of T cells, particularly CD4 1 T cells, is dictated by various stimulation signals including cytokines. Subpopulations of CD4 1 T cells are distinguished based on expression levels of surface protein markers, and on the identity of secreted cytokines. Differentiated T cells cooperate with other immune cells to either promote or suppress adaptive immune responses [18]. APCs, Antigen-presenting cells; MHC, major histocompatibility complex. Reprinted/adapted by permission from Elservier: Elsevier. Theranostic Bionanomaterials by Juan Du and Per Hydbring. Elsevier Ltd. All rights reserved. 2018.

3.1.4 Immunotherapy The main current immunotherapy approaches cover cancer vaccines, and various antibodies including checkpoint inhibitors. Examples of biological material used for cancer vaccine strategies are cancer proteins, dead tumor cells, pulsed DCs, and viral proteins. The majority of cancer vaccine strategies include tumor-derived antigens or adjuvants to promote the proliferation and activation of cytotoxic T cells. FDA approval has already been accomplished for the CTLA-4 antibody ipilimumab as well as for PD-1/PD-L1 antibodies, also referred to as checkpoint inhibitors. Tumor cells often display an elevated expression of PD-L1, which through its binding to the receptor PD-1 on T cells, suppresses the reactivity of T cells against tumor cells. Checkpoint inhibitors act by blocking this ligand to receptor interaction and thereby maintaining T cell activity. In addition, there are a number of alternative ways to promote the activity of the immune

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system in cancer patients, including isolation and expansion of tumor-infiltrating lymphocytes, gene transfer of the TCR and chimeric antigen receptor-expressing T cells (CAR-T). In addition to the checkpoint inhibitors, various antibodies have been used for immunotherapy including Rituximab, a CD20 antibody, and Trastuzumab (Herceptin), targeting the tyrosine kinase receptor Her2 [32,33].

3.1.5 Nanoparticles as Anticancer Drug-Delivery System Recently, there have been numerous reports of the utilization of nanoparticles as vehicles of drug delivery to tumors. However, the effect on the immune system is often neglected by the use of immune-compromised mouse models. Therefore, in order to truly assess the potential of using various nanoparticle systems for drug delivery, it is of absolute essence to use experimental models with integrated immune system components. Drug delivery to DCs: Zheng et al., who encapsulated the DNA-intercalating drug doxorubicin within silica nanoparticles, demonstrated an interesting example of such. The doxorubicin-formulated nanoparticles were subsequently used for assessment of maturation of DCs, release of cytokines, as well as of target delivery effect on triple-negative breast cancers. Nanoparticle-encapsulated doxorubicin improved delivery to the tumors along with increased cytokine release and elevated DC maturation [34]. An important follow up to this study would be a direct comparison of the antitumor effects comparing nanoparticleencapsulated cytostatic drugs with nanoparticles lacking the anticancer agent, or nanoparticles conjugated with immune cell targeted agents, especially since a variety of recent studies have only looked at the latter. Such studies include the investigation of anticancer effects as well as stimulatory effects on DCs from DC ligand DEC205 tagged polymeric nanoparticles [35], and the stimulatory effects on DCs from coating of CD40-targeting antibodies on nanoparticles [36]. Antigen delivery to DCs: A number of recent reports describe various nanoparticle systems utilized as antigen-delivering systems to immune system components, including DCs. Tu et al. reported on a system where microneedle arrays were coated with ovalbuminencapsulated silica nanoparticles for intradermal delivery [37]. Purwada et al. described a nanogel system able to self-assemble in the presence of protein. Once taken up by DCs, the nanogel released the formulated protein (e.g., ovalbumin, fibronectin, BSA) for DC processing and exhibition to T cells [38]. Other nanoparticle systems tested for DC delivery include aluminum nanoparticles and lipid nanoparticles. In particular when modified with polyethyleneimine, aluminum hydroxide nanoparticles display limited toxicity and high DC cytoplasm delivery efficacy, resulting in substantial tumor growth reduction and prolonged survival when tested in mouse tumor models [39]. An interesting example of a lipid nanoparticle system is the YSK lipid developed by the laboratory of Hideyoshi Harashima. The lipid was first optimized for delivery of different molecules, including cyclic-di-GMP, alpha-galactosylceramide, and siRNAs into various human immune cell lines, where it displayed superiority in comparison to RNAiMAX, mainly due to differences in levels of particle aggregation. The laboratory later tested the system for delivery to DCs in a mouse

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lymphoma model and siRNA-mediated silencing of the suppressor of cytokine signaling 1, resulting in a significant increase in cytokine expression with concomitant reduction of tumor growth [40 42]. Enhance immunity through targeting of TLRs: A more specific antitumor targeted approach to activate the immune system was demonstrated by Ruiz-de-Angulo et al., who trapped an antigen and a TLR9-agonist (CpG) in micelle nanoparticles. Following injection, the CpG-formulated nanoparticles took on a lymph node traveling route [43]. The immune stimulatory effect of CpG-formulated nanoparticles is well documented [44,45]. Other blunt immune stimulatory molecules include the TLR3-agonist polyinosinic polycytidylic acid, a double-stranded RNA (poly I:C) as well as TLR4 and TLR7 agonists. Their formulation into various nanoparticles has demonstrated immune stimulatory effects as well as antitumorigenic effects in both human and murine systems [46,47]. A particular example of a potent TLR4-agonist is lipopolysaccharide (LPS). This TLR4-agonist was formulated into a nanoparticle system for investigation of efficacy and tolerability in a mouse model of colorectal tumors resulting in high deposit of LPS particles to the tumors [48]. T cells: When it comes to activation of T cells, Mueller et al. demonstrated that nanoparticle formulation of tumor antigens resulted in an efficient targeting of B cells with a subsequent activation of CD4 1 T cells [49]. Moreover, Skwarczynski et al. conjugated dendrimer nanoparticles with epitopes of B cells leading to a dramatic antibody production with a subsequent CD4 1 T cell cytokine release [50]. Tang et al. designed cell surface-conjugated nanoparticles with an encapsulated IL-15 agonist. Delivery of this system to mouse tumor models resulted in a rather selective expansion of tumor-infiltrating T cells (up to 16-fold increase in T cell expansion). Further, the increase in T cells enabled a substantially higher tolerability for cytokine administration before any toxicity could be observed and significantly improved clearance of tumors by activated mouse T cells [51]. However, there is a need for a thorough comparison study investigating the antitumorigenic effects, along with the immune stimulatory effects of nanoparticles formulated with specific tumor antigen plus immune component agonist, nanoparticles formulated only with immune component agonist, and nanoparticles formulated with only tumor antigen in order to understand the contribution and impact of each component for immune system-mediated clearance of cancer cells. Adoptive cell transfer: A very elegant example of how to exploit the advantages of nanoparticles as drug-delivery tools was demonstrated by Huang et al. In order to ensure homing to the lymph nodes through preserved CD62L expression, primary T cells were extracted and cultured with supplemented rapamycin (an inhibitor of the mechanistic target of rapamycin and IL-2) followed by membrane coating with SN38 (a topoisomerase I inhibitor)-tagged nanoparticles. The engineered T cells were then implanted into the Eμ-myc Arf2/2 lymphoma mouse model where mouse survival was significantly prolonged compared to unformulated SN38 or SN38-tagged nanoparticles not tethered to the T cell membranes [52]. The depth of this study emphasizes the possible limitations of nanoparticles in anticancer therapeutics and immune system stimulation. In order to substantially halt an advanced cancer disease in its tracks, an ex vivo expanded immune cell component may be required in addition to drugformulated nanoparticles. Further, it would be of great interest to investigate how

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nonengineered ex vivo expanded T cells would perform in comparison to the nanoparticleengineered cells. Influence of nanoparticle size: There are a limited number of studies strictly looking at the influence of nanoparticle size for delivery efficacy and antitumor impact. Also, it should be emphasized that efficiency originating from particle size may look very different depending on other nanoparticle physical properties. Erdogar et al. investigated the antitumorigenic efficiency of cationic chitosan nanoparticles with encapsulated bacillus Calmette Guerin (BCG) in a rat model of bladder cancer. These authors reported on an optimal formulation size of 269 375 nm, resulting in up to 42% efficiency in encapsulating BCG. This nanoparticle size also proved optimal in promoting animal survival and reducing bladder tumor burden [53]. Formulating monoclonal antibodies into nanoparticles: The usage of monoclonal antibodies to specifically target cancer-associated proteins is one of the most sought after current approaches in immunotherapy. One such example is the monoclonal antibody TA99, raised against the gp75 antigen. Chu et al. coformulated TA99 with albumin-loaded nanoparticles followed by administration to mouse tumor models resulting in decreased tumor burden and prolonged mouse survival in comparison to nanoparticles without TA99 or unformulated TA99 [54].

3.2 Nanoparticles as Immunotherapy The notion that the administration of nanoparticles results in modulatory effects on the immune system responses is well established. However, is it always advisable to have an immune system stimulatory effect, and if not, how can you control this through your nanoparticle experimental design? If we start from the cells most likely to take up nanoparticles, DCs, targeting strategies may be tailored depending on the particular disease state where you either want to increase or decrease the maturation of DCs. Multiple studies have shown examples of how to increase DC maturation through different categories of nanoparticles [20,55 57]. In order to decrease DC maturation, it is a necessity to formulate an antiinflammatory compound within the nanoparticle. This could be particularly important when combating an overly active immune system leading to severe inflammation, an outcome often utilized and promoted by highly aggressive tumors. As an example of this, Barbosa et al. formulated the anti-inflammatory agent resveratrol into lipid nanoparticles. Nanoparticle formulated resveratrol blunted tumor necrosis factor potentiated DC activation to a higher degree compared to unformulated resveratrol [58]. When it comes to nondrug-formulated nanoparticles and cancer immunotherapy, there are multiple recent studies claiming antitumorigenic effects by an end output of increased number of CD8 1 T cells in mice, but through completely different nanoparticle structures [59,60]. For example, delivery of PLGA-based biodegradable nanoparticles to DCs blunted angiogenesis and tumor growth correlating with elevated numbers of CD8 1 T cells [61]. Nondrug-formulated nanoparticle targeting is not limited to DCs, but also myeloid cells are

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under investigation as targets in attempts to target tumors through biological components in their environment [62]. This field warrants more studies comparing the immune-stimulatory effects of different types of nanoparticles in the same experimental system in order to determine the optimal nanoparticle structure for tuned DC uptake and maturation.

3.2.1 Complementing Nanoparticle-Based Therapies Except for directly targeting the immune system, nanoparticle-based therapies can also be used as a complement therapy to directed immune system-targeting therapies in cancer. One such field is nanoparticle-based phototherapies, which include photothermal therapy (PTT) and photodynamic therapy, and of which there are numerous preclinical antitumor studies [63 65]. Using gold nanoparticles with a conjugated adaptor for specific targeting of melanomas, Zhang et al. could induce PTT-cell death specific for the tumor cells [66]. Kumar and Srivastava produced biocompatible and biodegradable IR 820-encapsulated polycaprolactone glycol chitosan nanoparticles. In combination with immunotherapy, these nanoparticles proved highly efficient to targeting metastatic breast cancer using a model of MCF-7 cells [67]. Another example of a complementing nanoparticle-based therapy is the utilization of hybrid particles such as the conjugation of nanoparticles to cytotoxic CD8 1 T cells, which were recently demonstrated as therapeutics against cancers related to the Epstein Barr virus [68]. The tLyp1 peptide hybrid nanoparticle displayed targeted efficacy against Treg cells proved by the boosted inhibitory effect of imatinib on Treg cells through blocked phosphorylation of STAT3 and STAT5. In the in vivo setting, administration of this hybrid particle resulted in reduced tumor burden and extended animal survival, correlating with decreased amounts of intratumoral Treg cells and increased numbers of intratumoral CD8 1 T cells [69].

3.3 Nanoparticles as Vaccines Against Cancer Due to the numerous reports demonstrating enhanced immune system function following nanoparticle administration, it is tempting to speculate for a future use of nanoparticles as vaccines against various forms of cancer. But what experimental evidence exists that nanoparticles would fulfill this function at a level which is advancing the field? DC vaccination therapy has been around as a concept for the last couple of decades and hundreds of clinical trials are registered or ongoing for DC vaccination therapy (www.clinicaltrials.gov). The potential benefit of adding nanoparticles to this area would be to further boost the activity of implanted DCs, something that has been suggested in multiple preclinical studies using mouse tumor models [59,70,71]. Further, there are numerous mouse model studies demonstrating a potent activation of the adaptive immune system, including increased antibody production, cytokine production, and activity of T cells, following nanoparticle-mediated vaccination [72,73]. Importantly, Fraser et al. demonstrated the ability of nanoparticles to establish an extended adaptive immunity following the encapsulation of a T cell memory chimeric

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MHC II antigen. Administration of MHC II nanoparticles proved successful all the way to nonhuman primates with immunity recorded during a time lapse of 119 days [74]. The repertoire of other examples of nanoparticle-mediated vaccination covers a variety of formulations, including protein- and DNA-based antigens. All studies report an increased adaptive immunity with activated CD4 1 and CD8 1 T cells, and increased cytokine production including interferon secretion. In addition, for the ability of nanoparticle-mediated vaccinations to block or halt cancer, Wang et al. demonstrated a complete resistance to lung tumor development following the preadministration of silica nanoparticles [75 78]. In particular, many studies have been conducted on the use of nanoparticle vaccines for melanomas. Saluja et al. demonstrated DC stimulation with a subsequent increase in cytostatic CD8 1 T cells following the administration of antigen-formulated nanoparticles with surfaceconjugated ligand targeting DCs [35]. Interestingly, mRNAs have also been tested as antigens in formulation with nanoparticles. Oberli et al. used such an approach resulting in the regression of melanoma tumors and a substantial prolongation of survival in mice. Effects were attributed to substantial increases in CD8 1 T cells [79]. Molino et al. developed a nonviral nanoparticle system for cancer vaccination using a linked pyruvate dehydrogenase E2 peptide. Remarkably, only one immunization with this system expanded the number of CD8 1 T cells reactive to the melanoma epitope by a level of 30 120 in the spleen and draining lymph nodes, respectively, and compared to nonconjugated peptide. When investigated in a mouse melanoma model, B16, E2-nanoparticles extended animal survival rates by 40% compared to control-treated animals [80]. Furthermore, using a similar mouse melanoma tumor model and a nanoparticle-based vaccine containing 500 antigen molecules on each nanoparticle, the majority of mice were protected from tumor initiation. In comparison, all animals in the control-treated group developed tumors. The strong effect on tumor blockage correlated with a substantial induction of CD8 1 T cells [81]. Moreover, in an identical melanoma tumor model, Lu reported biodegradable mesoporous silica nanoparticles as a delivery platform for cancer immunotherapy. In principle, functionalized silica nanoparticles were used as the foundation to deliver the antigen protein ovalbumin in concert with an agonist for TLR 9 to APCs. As with other studies mentioned above, administration of the silica nanoparticles resulted in massive expansion of CD8 1 T cells followed by reduced tumor growth [82]. The future will decide whether this tumor form is particularly suited for nanoparticle-mediated vaccination or whether melanomas are generally more accessible to immune system-enhancing therapeutics. Additional examples of nanoparticle-based vaccines include the surface decoration of attenuated bacteria on synthetic nanoparticles originating from plasmid DNA, encoding the vascular endothelial growth factor receptor 2 (VEGFR2), and cationic polymers. Oral in vivo administration of such nanoparticles resulted in blockage of tumor growth with a substantial activation of T cells and a concomitant increase in cytokine expression. Furthermore, it was evident that administration of VEGFR2 nanoparticles resulted in strong suppression of the tumor vasculature with increased tumor necrosis [83]. The encapsulation of CpG and epitope peptides into layered double hydroxide (LDH) nanoparticles resulted in substantially stronger CD8 1 T cell responses and blunted tumor growth compared with epitope-free

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LDH-based vaccines [84]. Finally, administration of nanoparticles loaded with another TLR agonist, imiquimod (R837), and surface-coated with tumor-specific antigens in therapeutic combination with checkpoint inhibitors displayed superiority in comparison to single-agent treatment [85]. It is currently not clear how all these examples of preclinical nanoparticle studies will add to the current clinical practice of DC vaccination. Furthermore, the broad range of nanoparticle structures and approaches for vaccine formulations makes a clinical transition complicated. There is definitely a need for comparative studies in nonhuman primates comparing multiple nanoparticle classes and formulations to reduce risks of possible complications or setbacks in clinical trials.

3.4 Nanoparticles as Diagnostics Interestingly, recent studies suggest that the applications of nanoparticles may extend beyond that of drug delivery and cancer vaccination. In fact, a few scientific reports propose the use of nanoparticles in diagnostics. So how could we utilize a synthetic particle for diagnostics? The key would be to modify the surface of the nanoparticle in a way that the particle would only react to tumor-specific material. Once a reaction takes place, additional chemical modifications are required to enable detection. This was exactly what Stark and Cheng performed with their nanocapsid platform engineered from a hepatitis E virus. Through chemical modification of the surface of the capsid, a tool was created for both tumor-specific detection and targeting [86]. Another example of nanoparticle-mediated diagnostics relies on the imaging of tumor-associated macrophages. Tumors rich in macrophage infiltration tend to accumulate iron oxide, resulting in a darker contrast in magnetic resonance imaging. A way to exploit this for diagnostics is to utilize administration of iron oxide nanoparticles. Iron oxide nanoparticles represent a noninvasive imaging approach and can be used to stage both primary and metastatic tumors through their inflammatory microenvironment. Furthermore, it is a promising tool for monitoring therapeutic responses to various treatments [87]. Kulkarni et al. developed an elegant reporter system where nanoparticles were engineered to codeliver chemotherapeutic drugs or immunotherapeutic drugs with a responding reporter element to tumors. If tumors were responding to the chemotherapy or immunotherapy in vivo, activation of caspase-3 would cleave a reporter sequence in a way that a fluorescent signal was relieved from its quencher. Utilizing this system, authors could differentiate between chemotherapy-sensitive and chemotherapy-resistant as well as immunotherapy-sensitive and immunotherapy-resistant tumors [88].

3.5 Challenges Although nanoparticle-based treatments have reached clinical trials, there are still a number of different challenges that need to be resolved before nanoparticle-based immunotherapeutics can compete with current FDA-approved immunotherapies [89 91]. In this closing

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section, we are discussing the biology behind these challenges as well as areas of improvement for the design of nanoparticle-based therapies.

3.5.1 Modulating Innate and Adaptive Immunity In this chapter we have described a number of recent studies on how to target the innate and adaptive immunity using nanoparticle-based therapies with the aim of combating cancer. As for the innate immune system, the results of these studies remind us of the efficiency and speed following innate immunity stimulation. Depending on what is targeted in innate immunity, responses can be more or less specific, as seen for TLR-targeting versus general DC activation. There is however still a lot we don’t understand when it comes to targeting innate immunity. The mechanistic outcomes are often unclear, making nanoparticlemediated targeting complicated. One should also remember that the durability of innate immunity is modest compared to adaptive immunity, which will likely dampen the soughtafter clinical effect. While TLR-targeting has shown great promise for clearance of infections, the potential for clinical cancer treatment is less obvious. Also, when it comes to nanoparticle-mediated vaccination of DCs, there is a lack of comprehensive literature. More studies are warranted in order to decipher the potential benefits compared to conventional DC vaccination. Activated T cells belonging to adaptive immunity have spurred an enormous interest as targets of various immune oncology studies. As mentioned earlier in this chapter, activated T cells include CD4 1 and CD8 1 cells, where the CD8 1 cells are also referred to as cytotoxic T cells. Although they hold great promise for the development of vaccines, the complex identity of CD4 1 T cells, where subtypes such as the Treg-cells are immunosuppressive, requires detailed top-quality studies. It is also important to remember that all nanoparticle deliveries to niches of APC residence are very likely to induce CD4 1 T cell responses since APCs will present the engulfed nanoparticle content through MHC-II complexes to CD4 1 T cells. In contrast to the activation of CD4 1 T cells, any cell carrying foreign material triggers CD8 1 T cell responses via exhibition through MHC-I protein complexes. Due to this, it is very challenging to produce a fast and specific CD8 1 T cell response from nanoparticles, although various approaches exist including functionalization of the nanoparticle surface with specific or multiple ligands.

3.5.2 Nanoparticle Characteristics In order for nanoparticles to interact with immune cells following systemic administration, it is essential that they possess a slow turnover time and that they remain in the circulation for an extended amount of time. Steric stabilization can be achieved by poly(ethylene glycol) [92 94]. However, targeting of solid tissues is still far from successful for most nanoparticles, although customized modifications for cell/tissue-specific delivery [95 97] become more common in order to reach a controlled immunity output. Still, our knowledge on nanoparticle surface properties, elasticity, shape, and sizes is immature. We know that all of these

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factors affect the target specificity and efficacy of different tissues [98,99], but due to the broad range of nanoparticle types, it is extremely challenging to define a “gold-standard” of nanoparticle characteristics. The field urgently needs a large comparative study taking into account the targeting effects of both the innate and adaptive immunity from a variety of nanoparticle classes with multiple particle types in each class. Selecting a system for a specific immune system component is currently based on limited literature and therefore in the end down to subjective bias.

3.6 Concluding Remarks The immune system is an immensely complex network where components from different parts communicate with each other. Due to its molecular characteristics, targeting of specific factors will eventually result in broader and less specific outcomes. In order to fully understand the impact of nanoparticle targeting of the immune system, studies need to be conducted in a way that monitoring of a larger panel of components becomes standard practice. For example, subgroups of CD4 1 T cells (e.g., Th1, Th2, and Th17), are rarely investigated in studies describing nanoparticle targeting of the immune system. Furthermore, biological mechanisms are often poorly investigated, relying on cytokine expression instead of modulating various components through genetic knockout systems. Also, the transition from mice to human clinical trials is cumbersome since specific parts of immunity, such as TLR innate immunity, are distinct between mice and humans. Despite all these obstacles, there are numerous nanoparticles currently under clinical investigation [100]. For the FDA-approved nanoparticles specifically, efforts have been focused on target site specificity and particle delivery efficacy [100]. Whether the vast number of studies investigating nanoparticles in various diseases will translate into modified and improved clinical practice in the near future is debatable. However, it is reasonable to believe that approaches combining ex vivo expansion of specific immune system components with drug-formulated nanoparticles could hold the key to success. Such approaches are in some ways similar to CAR-T cell therapy, which has already obtained FDA-approval in non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia (Kymriah—Novartis, Yescarta—Kite Pharma). CAR-T cell therapy has experienced limited efficacy in solid tumors, partly due to challenges to find tumor-specific antigens. Possibly, tethering of drugformulated nanoparticles to ex vivo expanded T cells may provide a specific benefit for solid tumors harboring mutations amenable to small-molecule targeting. Although unformulated small molecules can also target such tumors, the encapsulation into nanoparticles is likely to enhance target delivery.

References [1] O.C. Farokhzad, R. Langer, Nanomedicine: developing smarter therapeutic and diagnostic modalities, Adv. Drug Deliv. Rev. 58 (14) (2006) 1456 1459. [2] O.C. Farokhzad, R. Langer, Impact of nanotechnology on drug delivery, ACS Nano 3 (1) (2009) 16 20.

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69

[3] T. Sun, et al., Engineered nanoparticles for drug delivery in cancer therapy, Angew. Chem. Int. Ed. Engl. 53 (46) (2014) 12320 12364. [4] D. Peer, et al., Nanocarriers as an emerging platform for cancer therapy, Nat. Nanotechnol. 2 (12) (2007) 751 760. [5] J.M. Ageitos, J.A. Chuah, K. Numata, Design considerations for properties of nanocarriers on disposition and efficiency of drug and gene delivery, in: M. Braddock, (Ed.), Nanomedicines: Design, Delivery and Detection, 2016. [6] M.A. Dobrovolskaia, S.E. McNeil, Immunological properties of engineered nanomaterials, Nat. Nanotechnol. 2 (8) (2007) 469 478. [7] C. Petrarca, et al., Engineered metal based nanoparticles and innate immunity, Clin. Mol. Allergy 13 (1) (2015) 13. [8] A. Seth, D.B. Oh, Y.T. Lim, Nanomaterials for enhanced immunity as an innovative paradigm in nanomedicine, Nanomedicine (Lond.) 10 (6) (2015) 959 975. [9] T. Kobayashi, et al., Antitumor immunity by magnetic nanoparticle-mediated hyperthermia, Nanomedicine (Lond.) 9 (11) (2014) 1715 1726. [10] J.I. Andorko, K.L. Hess, C.M. Jewell, Harnessing biomaterials to engineer the lymph node microenvironment for immunity or tolerance, AAPS J. 17 (2) (2015) 323 338. [11] G. Sharma, et al., Nanoparticle based insulin delivery system: the next generation efficient therapy for Type 1 diabetes, J. Nanobiotechnol. 13 (2015) 74. [12] R. Toy, K. Roy, Engineering nanoparticles to overcome barriers to immunotherapy, Bioeng. Transl. Med. 1 (1) (2016) 47 62. [13] R. Medzhitov, C. Janeway Jr., Innate immunity, N. Engl. J. Med. 343 (5) (2000) 338 344. [14] C.A. Thaiss, et al., The microbiome and innate immunity, Nature 535 (7610) (2016) 65 74. [15] R. Warrington, et al., An introduction to immunology and immunopathology, Allergy Asthma Clin. Immunol. 7 (Suppl. 1) (2011) S1. [16] M.D. Vesely, et al., Natural innate and adaptive immunity to cancer, Annu. Rev. Immunol. 29 (2011) 235 271. [17] A. Iwasaki, R. Medzhitov, Regulation of adaptive immunity by the innate immune system, Science 327 (5963) (2010) 291 295. [18] J. Du, et al., Nanoparticles for immune system targeting, Drug Discov. Today 22 (9) (2017) 1295 1301. [19] T. Frenz, et al., Antigen presenting cell-selective drug delivery by glycan-decorated nanocarriers, Eur. J. Pharm. Biopharm. 95 (Pt A) (2015) 13 17. [20] C. Lonez, et al., Cationic lipid nanocarriers activate Toll-like receptor 2 and NLRP3 inflammasome pathways, Nanomedicine 10 (4) (2014) 775 782. [21] B. Sun, et al., Engineering an effective immune adjuvant by designed control of shape and crystallinity of aluminum oxyhydroxide nanoparticles, ACS Nano 7 (12) (2013) 10834 10849. [22] Y.T. Lee, et al., Respiratory syncytial virus-like nanoparticle vaccination induces long-term protection without pulmonary disease by modulating cytokines and T-cells partially through alveolar macrophages, Int. J. Nanomed. 10 (2015) 4491 4505. [23] M.T. Stephan, et al., Synapse-directed delivery of immunomodulators using T-cell-conjugated nanoparticles, Biomaterials 33 (23) (2012) 5776 5787. [24] X. Wang, et al., Rod-shaped and fluorine-substituted hydroxyapatite free of molecular immunopotentiators stimulates anti-cancer immunity in vivo, Chem. Commun. (Camb.) 52 (44) (2016) 7078 7081. [25] J. Wang, et al., The changes of T lymphocytes and cytokines in ICR mice fed with Fe3O4 magnetic nanoparticles, Int. J. Nanomed. 6 (2011) 605 610.

70

THERANOSTIC BIONANOMATERIALS

[26] S. Lee, et al., The comparative immunotoxicity of mesoporous silica nanoparticles and colloidal silica nanoparticles in mice, Int. J. Nanomed. 8 (2013) 147 158. [27] J. Borst, et al., CD4(1) T cell help in cancer immunology and immunotherapy, Nat. Rev. Immunol. 18 (10) (2018) 635 647. [28] J. Matsuzaki, et al., Direct tumor recognition by a human CD4(1) T-cell subset potently mediates tumor growth inhibition and orchestrates anti-tumor immune responses, Sci. Rep. 5 (2015) 14896. [29] G. Plitas, A.Y. Rudensky, Regulatory T cells: differentiation and function, Cancer Immunol. Res. 4 (9) (2016) 721 725. [30] M. Frydrychowicz, et al., The dual role of treg in cancer, Scand. J. Immunol. 86 (6) (2017) 436 443. [31] W.L. Byrne, et al., Targeting regulatory T cells in cancer, Cancer Res. 71 (22) (2011) 6915 6920. [32] S.M. Chiavenna, J.P. Jaworski, A. Vendrell, State of the art in anti-cancer mAbs, J. Biomed. Sci. 24 (1) (2017) 15. [33] C.T.T. Hagan, Y.B. Medik, A.Z. Wang, Nanotechnology approaches to improving cancer immunotherapy, Adv. Cancer Res. 139 (2018) 35 56. [34] D.W. Zheng, et al., Highly integrated nano-platform for breaking the barrier between chemotherapy and immunotherapy, Nano Lett. 16 (7) (2016) 4341 4347. [35] S.S. Saluja, et al., Targeting human dendritic cells via DEC-205 using PLGA nanoparticles leads to enhanced cross-presentation of a melanoma-associated antigen, Int. J. Nanomed. 9 (2014) 5231 5246. [36] R.A. Rosalia, et al., CD40-targeted dendritic cell delivery of PLGA-nanoparticle vaccines induce potent anti-tumor responses, Biomaterials 40 (2015) 88 97. [37] J. Tu, et al., Mesoporous silica nanoparticle-coated microneedle arrays for intradermal antigen delivery, Pharm. Res. 34 (8) (2017) 1693 1706. [38] A. Purwada, et al., Self-assembly protein nanogels for safer cancer immunotherapy, Adv. Healthc. Mater. 5 (12) (2016) 1413 1419. [39] H. Dong, et al., Polyethyleneimine modification of aluminum hydroxide nanoparticle enhances antigen transportation and cross-presentation of dendritic cells, Int. J. Nanomed. 13 (2018) 3353 3365. [40] T. Nakamura, et al., Small-sized, stable lipid nanoparticle for the efficient delivery of siRNA to human immune cell lines, Sci. Rep. 6 (2016) 37849. [41] Y. Sato, et al., Development of a multifunctional envelope-type nano device and its application to nanomedicine, J. Control. Release 244 (Pt B) (2016) 194 204. [42] S. Warashina, et al., A lipid nanoparticle for the efficient delivery of siRNA to dendritic cells, J. Control. Release 225 (2016) 183 191. [43] A. Ruiz-de-Angulo, et al., Microdosed lipid-coated (67)Ga-magnetite enhances antigen-specific immunity by image tracked delivery of antigen and CpG to lymph nodes, ACS Nano 10 (1) (2016) 1602 1618. [44] M. Wei, et al., Polyvalent immunostimulatory nanoagents with self-assembled CpG oligonucleotideconjugated gold nanoparticles, Angew. Chem. Int. Ed. Engl. 51 (5) (2012) 1202 1206. [45] J.H. Kim, et al., Multifunctional hybrid nanoconjugates for efficient in vivo delivery of immunomodulating oligonucleotides and enhanced antitumor immunity, Angew. Chem. Int. Ed. Engl. 51 (38) (2012) 9670 9673. [46] A. Ultimo, et al., Targeting innate immunity with dsRNA-conjugated mesoporous silica nanoparticles promotes antitumor effects on breast cancer cells, Chemistry 22 (5) (2016) 1582 1586. [47] C.B. Fox, et al., A nanoliposome delivery system to synergistically trigger TLR4 AND TLR7, J. Nanobiotechnol. 12 (2014) 17. [48] M.A. Shetab Boushehri, et al., A nanoparticle-based approach to improve the outcome of cancer active immunotherapy with lipopolysaccharides, Drug Deliv. 25 (1) (2018) 1414 1425.

Chapter 3 • Nanoparticle Interaction With Immune Cells

71

[49] S.N. Mueller, S. Tian, J.M. DeSimone, Rapid and persistent delivery of antigen by lymph node targeting PRINT nanoparticle vaccine carrier to promote humoral immunity, Mol. Pharm. 12 (5) (2015) 1356 1365. [50] M. Skwarczynski, et al., Polyacrylate dendrimer nanoparticles: a self-adjuvanting vaccine delivery system, Angew. Chem. Int. Ed. Engl. 49 (33) (2010) 5742 5745. [51] L. Tang, et al., Enhancing T cell therapy through TCR-signaling-responsive nanoparticle drug delivery, Nat. Biotechnol. 36 (8) (2018) 707 716. [52] B. Huang, et al., Active targeting of chemotherapy to disseminated tumors using nanoparticle-carrying T cells, Sci. Transl. Med. 7 (291) (2015) 291ra94. [53] N. Erdogar, et al., Antitumor efficacy of bacillus Calmette-Guerin loaded cationic nanoparticles for intravesical immunotherapy of bladder tumor induced rat model, J. Nanosci. Nanotechnol. 15 (12) (2015) 10156 10164. [54] D. Chu, et al., Nanoparticle targeting of neutrophils for improved cancer immunotherapy, Adv. Healthc. Mater. 5 (9) (2016) 1088 1093. [55] A. Yeste, et al., Tolerogenic nanoparticles inhibit T cell-mediated autoimmunity through SOCS2, Sci. Signal. 9 (433) (2016) ra61. [56] M. Ghoneum, A. Ghoneum, J. Gimzewski, Nanodiamond and nanoplatinum liquid, DPV576, activates human monocyte-derived dendritic cells in vitro, Anticancer Res. 30 (10) (2010) 4075 4079. [57] S. De Koker, et al., Engineering polymer hydrogel nanoparticles for lymph node-targeted delivery, Angew. Chem. Int. Ed. Engl. 55 (4) (2016) 1334 1339. [58] J.P. Barbosa, et al., Nanostructured lipid carriers loaded with resveratrol modulate human dendritic cells, Int. J. Nanomed. 11 (2016) 3501 3516. [59] M.E. Lebel, et al., Potentiating cancer immunotherapy using papaya mosaic virus-derived nanoparticles, Nano Lett. 16 (3) (2016) 1826 1832. [60] R. Suzuki, et al., Tumor growth suppression by the combination of nanobubbles and ultrasound, Cancer Sci. 107 (3) (2016) 217 223. [61] R.A. Kokate, et al., Rationalizing the use of functionalized poly-lactic-co-glycolic acid nanoparticles for dendritic cell-based targeted anticancer therapy, Nanomedicine (Lond.) 11 (5) (2016) 479 494. [62] A. Tuettenberg, K. Steinbrink, D. Schuppan, Myeloid cells as orchestrators of the tumor microenvironment: novel targets for nanoparticular cancer therapy, Nanomedicine (Lond.) 11 (20) (2016) 2735 2751. [63] X. Hou, et al., Nanoparticle-based photothermal and photodynamic immunotherapy for tumor treatment, Int. J. Cancer 143 (12) (2018) 3050 3060. [64] S.K. Rajendrakumar, et al., Nanoparticle-based phototriggered cancer immunotherapy and its domino effect in the tumor microenvironment, Biomacromolecules 19 (6) (2018) 1869 1887. [65] T. Yata, et al., DNA nanotechnology-based composite-type gold nanoparticle-immunostimulatory DNA hydrogel for tumor photothermal immunotherapy, Biomaterials 146 (2017) 136 145. [66] Y. Zhang, et al., Temperature-dependent cell death patterns induced by functionalized gold nanoparticle photothermal therapy in melanoma cells, Sci. Rep. 8 (1) (2018) 8720. [67] P. Kumar, R. Srivastava, IR 820 dye encapsulated in polycaprolactone glycol chitosan: poloxamer blend nanoparticles for photo immunotherapy for breast cancer, Mater. Sci. Eng. C: Mater. Biol. Appl. 57 (2015) 321 327. [68] R.A. Burga, et al., Conjugating Prussian blue nanoparticles onto antigen-specific T cells as a combined nanoimmunotherapy, Nanomedicine (Lond.) 11 (14) (2016) 1759 1767. [69] W. Ou, et al., Regulatory T cell-targeted hybrid nanoparticles combined with immuno-checkpoint blockage for cancer immunotherapy, J. Control. Release 281 (2018) 84 96.

72

THERANOSTIC BIONANOMATERIALS

[70] S.Y. Kim, et al., Multivalent polymer nanocomplex targeting endosomal receptor of immune cells for enhanced antitumor and systemic memory response, Angew. Chem. Int. Ed. Engl. 54 (28) (2015) 8139 8143. [71] A.V. Li, et al., Generation of effector memory T cell-based mucosal and systemic immunity with pulmonary nanoparticle vaccination, Sci. Transl. Med. 5 (204) (2013) 204ra130. [72] Y.W. Noh, et al., Polymer nanomicelles for efficient mucus delivery and antigen-specific high mucosal immunity, Angew. Chem. Int. Ed. Engl. 52 (30) (2013) 7684 7689. [73] T. Hunsawong, et al., A novel dengue virus serotype-2 nanovaccine induces robust humoral and cellmediated immunity in mice, Vaccine 33 (14) (2015) 1702 1710. [74] C.C. Fraser, et al., Generation of a universal CD4 memory T cell recall peptide effective in humans, mice and non-human primates, Vaccine 32 (24) (2014) 2896 2903. [75] X. Wang, et al., Stimulation of in vivo antitumor immunity with hollow mesoporous silica nanospheres, Angew. Chem. Int. Ed. Engl. 55 (5) (2016) 1899 1903. [76] W. Gao, et al., Modulating antibacterial immunity via bacterial membrane-coated nanoparticles, Nano Lett. 15 (2) (2015) 1403 1409. [77] B. Li, et al., Improved proliferation of antigen-specific cytolytic T lymphocytes using a multimodal nanovaccine, Int. J. Nanomed. 11 (2016) 6103 6121. [78] W. Ji, et al., Poly(2-aminoethyl methacrylate) with well-defined chain length for DNA vaccine delivery to dendritic cells, Biomacromolecules 12 (12) (2011) 4373 4385. [79] M.A.R. Oberli, A.M. Reichmuth, J.R. Dorkin, M.J. Mitchell, O.S. Fenton, A. Jakienec, et al., Lipid nanoparticle assisted mRNA delivery for potent cancer immunotherapy, Nano Lett. 17 (3) (2017) 1326 1335. [80] N.M. Molino, et al., Viral-mimicking protein nanoparticle vaccine for eliciting anti-tumor responses, Biomaterials 86 (2016) 83 91. [81] K. Wang, et al., “Minimalist” nanovaccine constituted from near whole antigen for cancer immunotherapy, ACS Nano 12 (7) (2018) 6398 6409. [82] Y. Lu, et al., Glutathione-depletion mesoporous organosilica nanoparticles as a self-adjuvant and codelivery platform for enhanced cancer immunotherapy, Biomaterials 175 (2018) 82 92. [83] Q. Hu, et al., Engineering nanoparticle-coated bacteria as oral DNA vaccines for cancer immunotherapy, Nano Lett. 15 (4) (2015) 2732 2739. [84] L.X. Zhang, et al., Efficient co-delivery of neo-epitopes using dispersion-stable layered double hydroxide nanoparticles for enhanced melanoma immunotherapy, Biomaterials 174 (2018) 54 66. [85] R. Yang, et al., Cancer cell membrane-coated adjuvant nanoparticles with mannose modification for effective anticancer vaccination, ACS Nano 12 (6) (2018) 5121 5129. [86] M. Stark, R.H. Cheng, Surface modulatable nanocapsids for targeting and tracking toward nanotheranostic delivery, Pharm. Pat. Anal. 5 (5) (2016) 307 317. [87] N.J. Serkova, Nanoparticle-based magnetic resonance imaging on tumor-associated macrophages and inflammation, Front. Immunol. 8 (2017) 590. [88] A. Kulkarni, et al., Reporter nanoparticle that monitors its anticancer efficacy in real time, Proc. Natl. Acad. Sci. U.S.A. 113 (15) (2016) E2104 E2113. [89] A.J. Grippin, E.J. Sayour, D.A. Mitchell, Translational nanoparticle engineering for cancer vaccines, Oncoimmunology 6 (10) (2017) e1290036. [90] E. Hong, M.A. Dobrovolskaia, Addressing barriers to effective cancer immunotherapy with nanotechnology: achievements, challenges, and roadmap to the next generation of nanoimmunotherapeutics, Adv. Drug Deliv. Rev. (2018). Available from: https://doi.org/10.1016/j.addr.2018.01.005. [91] S. Sengupta, Cancer nanomedicine: lessons for immuno-oncology, Trends Cancer 3 (8) (2017) 551 560.

Chapter 3 • Nanoparticle Interaction With Immune Cells

73

[92] Y. Wang, et al., Comparison study of gold nanohexapods, nanorods, and nanocages for photothermal cancer treatment, ACS Nano 7 (3) (2013) 2068 2077. [93] A. Albanese, P.S. Tang, W.C. Chan, The effect of nanoparticle size, shape, and surface chemistry on biological systems, Annu. Rev. Biomed. Eng. 14 (2012) 1 16. [94] A.C. Anselmo, et al., Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting, ACS Nano 9 (3) (2015) 3169 3177. [95] S. Mura, J. Nicolas, P. Couvreur, Stimuli-responsive nanocarriers for drug delivery, Nat. Mater. 12 (11) (2013) 991 1003. [96] C. Alvarez-Lorenzo, A. Concheiro, Smart drug delivery systems: from fundamentals to the clinic, Chem. Commun. (Camb.) 50 (58) (2014) 7743 7765. [97] E.J. Kwon, J.H. Lo, S.N. Bhatia, Smart nanosystems: bio-inspired technologies that interact with the host environment, Proc. Natl. Acad. Sci. U.S.A. 112 (47) (2015) 14460 14466. [98] M. Ferrari, Cancer nanotechnology: opportunities and challenges, Nat. Rev. Cancer 5 (3) (2005) 161 171. [99] R.A. Petros, J.M. DeSimone, Strategies in the design of nanoparticles for therapeutic applications, Nat. Rev. Drug Discov. 9 (8) (2010) 615 627. [100] D. Bobo, et al., Nanoparticle-based medicines: a review of FDA-approved materials and clinical trials to date, Pharm. Res. 33 (10) (2016) 2373 2387.

4 Clinical Translation of Nanomaterials Han Liu1,2, Peter van Nooten1,2, Lianfu Deng1, Wenguo Cui1 1

SHANGHAI INSTITUT E OF TRAUMATOLOG Y AND ORTHOPAEDICS, SHANGHAI KEY LABORAT ORY FOR PREV ENT ION AND TREATM ENT O F B ONE AND JOINT DISEASE S, RUIJIN HOSPITAL, SHANGHAI J IAO TONG UNIVERSITY SCH OOL OF MEDICINE, SHANGHAI, P.R. C HI NA 2 DE PARTMENT OF NANOTECHNOLOGY ENGINEERING, UNIVERSITY OF WATERLOO, WATERL OO, ON, C ANADA

Since Feynman’s inspiring speech on the “plenty of room at the bottom,” in merely half a century, the field of nanotechnology has grown and flourished at a dazzling speed. Marching into the 21st century, more than 60 countries had established specialized institutes and initiated national projects in nanotechnology to prompt further developments in the area [1]. In 2004, the US federal funding for nanotechnology was $961 million; and the European Union budget allocated for nanotechnology research reached an astonishing h3.5 billion in 2007 [2]. More recently, in 2015, the global nanotechnology market was projected to reach $30.4 billion, with many nanotechnology products already commercialized and earning revenues for the public sector; many believe nanotechnology will exceed the impact of the Industrial Revolution on our society [3]. As a result of decades of unwavering research endeavors and substantial funds, appropriately, an impressive amount of close to 50,000 nano-related patent applications were approved and granted in the past 20 years [4]. Nanotechnology has been transforming virtually every aspect of our life: in 2008, a nano-switch made of graphene transistors was designed to replace traditional silicon [5]; in 2015, heat-triggered liposomes, hailed as the “holy grail of nanomedicine” to conquer cancer, improved the survival rates in the initial animal trials successfully [6]; more recently, the Nobel Prize in Chemistry was awarded to a nano-bot developed to be a miniaturized surgeon of the future [6]. Covering all fields that nanotechnology has revolutionized renders an immense task. Thus we will focus on nanomaterials with potential or already commercialized applications in medicine, more specifically, the clinical translation of these nanomaterials. The prefix “nano,” Greek for “dwarf,” is a length measurement unit for one-billionth of a meter, approximately a hundred-thousandth the diameter of human hair [7]. Thus nanotechnology in medicine generally refers to the design, synthesis, and characterization of molecules with atomic precision for purposes including but not limited to preventing enzymatic degradation, controlling triggered release, directing cellular behaviors, etc. This summative review highlights the advances achieved in the state-of-the-art nanomaterials for diagnostic, Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00004-3 © 2019 Elsevier Inc. All rights reserved.

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therapeutic, and tissue regeneration purposes, from three main categories: inorganic nanomaterials, polymeric nanomaterials, and lipid-based nanomaterials.

4.1 Diagnostics The cornerstone of diagnostic nanomaterials was laid by Leland C. Clark with the successful fabrication of enzyme electrodes as biosensors in 1962. Since then, point-of-care biosensors have gained popularity over laboratory testing rapidly, led by companies such as Nova Biomedical, Medtronic Diabetes, and Abbott Point of Care [8]. Today, a wide variety of new diagnostic nanomaterials has been developed using quantum dots (QDs), carbon nanotubes (CNTs), and inorganic nanoparticles (INPs); the primary focus of this section is on the clinical translation of the aforelisted diagnostic nanomaterials.

4.1.1 Carbon Nanotubes CNTs are easily the most well-known nanomaterials from the field of nanotechnology. First discovered by Lijima in 1991, CNTs are thin sheets of benzene ring carbons rolled up into a hollow tubular structure, and can be roughly classified into two categories based on the number of concentric graphene layers contained: single-walled CNTs (SWCNTs) and multiwalled CNTs (MWCNTs) [9]. Due to their unique physicochemical properties, CNTs are believed to have a vast number of potential applications in diverse fields from electronic systems as the “heir to silicon’s throne” to cancer therapy as bio-theranostics [10]; they have even inspired a few neat futuristic architectural designs [11]. Their extraordinary thermal, optical, mechanical, and electrical properties render CNTs particularly desirable for the clinical translation of biomedicine. It has been well established that CNTs can pass through the membrane and internalize within the cell, which makes them promising therapeutic agents. Plenty of comprehensive review articles can be found on the subject of CNT-based therapy in literature databases [12 14]. Many companies have received regulatory approval to enter the clinical trial stage for CNT therapeutics, including Ensysce Biosciences Inc.’s siRNA delivering SWCNTs for cancer treatment and similarly Calando Pharmaceuticals Inc.’s IT-101, as well as Teg BioSciences Corporation’s TBP project for Parkinson’s disease [15]. In comparison, research on the diagnostic functionalities of CNTs has grown rapidly. The engineering possibilities of delivering a wide array of imaging agents and tracers have been carefully explored in the past decade [16]. The ability to detect cancer at its early stage is imperative to set prompt treatment in motion and prevent future metastasis. Without timely resection operation, patients then have to resort to chemotherapy, which lacks selectivity and subjects them to many undesirable side effects. Firstly, for feasible clinical applications, functionalization is essential for pristine CNTs to be soluble in physiological conditions. By covalently linking organic moieties to CNTs, numerous synthetic approaches in this area have been proven successful, and exhaustive reviews have be published [17]. In general, CNT tips are usually covalently modified by strong oxidizing agents, which also help eliminate impurities; CNT sidewalls are usually

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covalently modified by cycloaddition or radical reactions [18]. Furthermore, functionalization can mitigate the intrinsically toxic effects at CNT surfaces; for example, in vitro studies had indicated that SWCNTs functionalized by covalent phenyl-SO3H or phenyl-(COOH)2 groups demonstrated less cytotoxicity than the aqueous dispersions of pristine SWCNT stabilized with 1% Pluronic F108 surfactant [19]. Gold plating CNTs has also been reported to address this problem successfully [20]. The clinical relevance of CNTs has long been a hot area of debate among researchers; many factors can influence the toxicity of CNTs including the structural defects, single-walled or multiwalled, region of accumulation, etc. [21 26]. Due to the complexity of CNT toxicity, it will not be the primary focus of this review. CNTs are inherently excellent diagnostic matrices as their internal hollow center can encapsulate imaging agents and the great potential of their physiological properties can be harnessed for applications. For instance, metal halides, such as CuBr and NaI, can be sealed inside SWCNTs to create high-density radio-emitting crystals for ultrasensitive imaging [27]; upon exploiting the dependence of SWCNT conductance on the molecule-specific surface absorption and electrostatic charge in the surrounding environment, miniature sensors were built for biomolecule detection under physiological conditions, especially the level of serum proteins such as markers and antibodies after therapeutic intervention or vaccinations [28]. In recent years, CNTs have been used as a performance-enhancing contrasting agent in many imaging techniques, as shown in Fig. 4 1. Magnetic resonance imaging (MRI) is one of the most widely adopted methodologies in clinics today for its noninvasive nature and real-time monitoring. Unfortunately, conventional techniques like MRI lack spatial resolution and are not able to detect morphologic changes that only become obvious in later stages [29]. Gd31 complexes and

FIGURE 4–1 Imaging applications of carbon nanotubes in Raman imaging, photoacoustic imaging, MRI, NIR-II imaging, and PET imaging. MRI, magnetic resonance imaging.

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superparamagnetic iron-oxide (SPIO) nanoparticles (NPs) are the most commonly used positive and negative contrast agents used to furnish extra details in MRI imaging. Unfortunately, they both fall short in terms of selective binding and Gd31 suffers from low relaxivity and thus low efficiency. CNTs have been found useful to address these issues. Recently, MRI Gd31 enhancing contrast agent was developed by noncovalently decorating the outer surface of MWCNTs with amphiphilic gadolinium chelate [30]; SPIO enhancing cancer-targeting contrast agent was developed by folic acid-conjugated MWCNT/SPIO hybrid [31]. Fluorescence is another powerful imaging methodology due to its ability to resolve features down to the diffraction limit and beyond. However, the value of fluorescence in clinical applications is diminished by its compromised penetration depth. The past decade has witnessed productive research efforts in developing and implementing sensor probes in near-infrared (NIR) regions—a “biological transparency window” where the depth-induced attenuation of signal is minimal [32]. The NIR photoluminescence intrinsic to SWCNTs renders them a natural and strong candidate for fluorescent contrast agents. Further, it has been proven to possess an advantageous emission range of 1100 1140 nm compared to 800 nm and SWCNT-based diagnostics demonstrated remarkably high resolution and deep penetration [33]. A note on the issue of depth-induced resolution loss, photoacoustic imaging is an emerging technique worth mentioning. Its working principle relies on the conversion of light into ultrasound, which is subsequently detected and measured externally. The advent of photoacoustic imaging brought hope to overcome the depth-related resolution loss frequently encountered in optical imagings. However, since not all tumors manifest an endogenous contrast, Zerda et al. reported on a contrasting agent, dye-enhanced cyclicpeptide-conjugated SWCNTs that bound with tumor angiogenesis-associated integrins; the contrasting agent achieved a truly remarkable sub-nM sensitivity and a submillimeter spatial resolution [34]. Successful cases of CNT-based nano-hybrids as multimodal cellular imaging agents have also been reported in literature. For example, the magnetic and fluorescent imaging agent are highly practical for locating and removing tumors in surgery. When SPIO NPs and NIR fluorescent CdTe QDs were sequentially coupled on CNTs, the unique physicochemical property of CNTs allowed SPIO NPs to lodge orderly and mono-directionally along CNT surfaces, which in turn enhanced the overall magnetic effects [35].

4.1.2 Nanoparticles INPs refer to metallic or metal oxide particles with at least one dimension within the 100 nm range. Contrary to popular belief, the study of INPs is not a modern invention; in fact, the medical use of gold NPs can be traced back to ancient Chinese medicine and INPs have received revived research interest to meet the challenging requirements of modern imaging [36]. For example, fluorophores are signal transduction tools used to signal a biomolecule recognition event during the bio-imaging process. Although conventional organic fluorophores are easy to use; limited by their binding ability to target molecules, they tend to produce weak signals and require postliminary amplification. In this light, INPs were developed and implemented fluorescence signal enhancement agents. INPs are designed to entrap a

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large quantity of dye molecules; and for many, they rely on precise tuning of their physicochemical parameters. When necessary, the surfaces of these NPs can also be subject to bioconjugation of stealth or targeting ligands for further amplification [37]. Silica, or silicon dioxide (SiO2), is one of the most extensively researched NPs for diagnostic purposes. As of January 2018, the time when this review was written, a search of “silica imaging NP” in the scholar literature database yielded more than 600,000 results, second only to gold NPs, likely due to its other applications such as catalysts; still, silica NPs outnumbered the titania, alumina, and SPIO NP counterparts combined. Silica NPs are biocompatible and have been demonstrated to be a versatile tool for detection of bacteria, DNA, and cancer [38 40]. To a certain extent, the success of silica NPs as detection agents should be attributed to their advanced synthesis technique—the Stober method—which first produced a monodisperse suspension of silica spheres in the colloidal size range successfully [41]. In addition, the superparamagnetic property allows silica NPs to manipulate trace amounts of bio-analytes from complex biological samples [42]. For instance, Deng et al. demonstrated selective binding and capture of phosphoproteins from complex protein mixtures, such as milk and egg, using guanidinium functionalized superparamagnetic silica [43]. In 2010, Cornell dots, also known as “C-dots,” marked the first silica-based diagnostic device approved for stage I human clinical trial by the Food and Drug Administration (FDA) [44]; the approval supports the confidence held for the clinical translation of silica NPs. In their first in vivo study, C-dots demonstrated utility in real-time intraoperative detection, as well as imaging of nodal metastases and lymphatic drainage [37]. It is worth noting that successful implementation of silica NPs requires restricting the size of silica NPs to the “goldilocks zone,” where NPs are not too small to entrap enough dye molecules for sufficiently bright fluorescent signals, but not so large that they block binding sites in close proximity [45]. Balancing the trade-off between signal intensity and binding success is crucial for performance optimization. Although having experienced preliminary success, silica NPs still faces many challenges awaiting resolution, such as premature dye leakage and unclear long-term in vivo fate [46]. Besides their diagnostic excellence in detection and imaging, silica NPs have also been developed as drug-delivery carriers for therapeutic purposes [44,47]. Gold NPs (AuNPs) are the most extensively researched noble metal NP and have been used in clinical translation for their unique optical and photothermal properties [37]. When a gold metal particle is exposed to a photon’s oscillating electromagnetic field, a collective oscillation of free electrons in the particle is induced, creating a dipole oscillation along the light’s direction [48], as shown in Fig. 4 2A. The amplitude of this oscillation reaches a peak at a specific frequency, known as surface plasmon resonance (SPR), as demonstrated in Fig. 4 2A. Tunable optical properties of gold nanorods are possible by changing the aspect ratios Fig. 4 2B. Incident light is strongly absorbed and scattered, orders of magnitude stronger than absorbing dye molecules and emitting fluorescent molecules [49]. Size, shape, structure, and composition all affect the SPR frequency and ratio of photon absorption to scattering. AuNPs have many potential opportunities in the fields of drug delivery, diagnostics, and therapeutics using this unique property as well as low cell toxicity [50]. AuNP shape and composition

FIGURE 4–2 (A) Schematic illustration of AuNP oscillation induced by photon’s electromagnetic field, and the relationship between the oscillation amplitude and frequency of AuNPs. (B) Tunable optical properties of gold nanorods by changing the aspect ratios. Gold nanorods of different aspect ratios exhibit different dimensions as seen by TEM (I), in different color (II) and different SPR wavelength (III).

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have developed to include geometries such as nanorods, nanoshells, nanostars, and nanocages, because they provide variable absorption peaks [51]. Gold nanorods have two SPR bands wherein the longitudinal band is located close to the NIR region, ideal for tissue penetration. By experimentally increasing the length to width aspect ratio of the nanorod, it can be shifted further toward the NIR region. Gold nanoshells can be redshifted by decreasing shell thickness and gold nanocages can be redshifted by controlling auric acid solution volume during fabrication. AuNPs also can convert light into heat through complex nonradiative processes [48,52], giving rise to photothermal therapy and tumor ablation technologies [53]. Through the enhanced permeability and retention effect, AuNPs accumulate passively in tumors due to their leaky vasculature. AuNPs can also be easily functionalized, via goldthiol bioconjugation chemistry, with passivating/therapeutic agents or targeting ligands to enhance biocompatibility. By fine-tuning optical properties to obtain maximum NIR absorption, tumors can be subjected to NIR via lasers that are irreversibly damaged by heat generated from the AuNPs. Currently in clinical trials, AuroLase is a silica gold nanoshell coated with polyethylene glycol (PEG) for thermal ablation of solid prostate tumors [54]. The silica makes up the dielectric core, while the thin gold shell absorbs NIR light. This selective therapy is advantageous over traditional therapies as it avoids systemic side effects, limiting damage to living tissue. With several applications already FDA-approved and many others in clinical studies, iron-oxide NPs (IONPs) have huge potential in imaging, diagnostics, and therapeutics [37,55]. Made up of magnetite NPs (Fe3O4) and its oxide form maghemite (γ-Fe3O4) along with an organic coating for biocompatibility, IONPs are useful because of (1) negligible cytotoxicity, (2) tunable magnetic properties, (3) controllable size and surface modification, and (4) MRI applications as contrast agent. When IONPs are reduced in size to the sub-100 nm range they exhibit superparamagnetic properties and SPIO NPs are achieved. Primarily studied as a contrast agent for MRI imaging, SPIO NPs have no magnetization in the absence of an external magnetic field but strong magnetization in the presence of one, capable of causing microscopic field inhomogeneity and dephasing protons [56]. As a result, T2 relaxation times are reduced and signal intensity of surrounding tissue is decreased in T2-weighted MR images. Particle size is the greatest determining factor in the IONP’s characteristics and they are thus grouped into three categories depending on whether their diameters are several micrometers, hundreds of nanometers, or less than 50 nm, respectively called micrometersized paramagnetic iron oxide, SPIO, and ultrasmall superparamagnetic iron oxide (USPIO). Additionally, these NPs also heat up in an alternating electric field, leading to applications in hyperthermia treatment [57] and thermal ablation of solid tumors [58]. Ferumoxytol, USPIOs coated with polyglucose sorbitol carboxymethylether, is FDA-approved as a treatment for iron deficiency in adults with chronic kidney disease. As the only application of IONPs currently approved by the FDA, it is also in clinical trials for many types of MRI imaging of cancer and diseases and other forms of anemia. For example, a recent study was able to visualize the progression of type 1 diabetes by detecting increased ferumoxytol concentrations in pancreatic lesions of patients with type 1 diabetes compared to control groups in mouse models and a pilot human study [59]. Another preclinical study uses ferumoxytol

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along with drug molecules heparin and protamine to create nanocomplexes capable of labeling stem or immune cells for MRI imaging [60]. Further studies are testing ferumoxytol as an applicant for targeted drug delivery by using weak electrostatic reactions to attach drugs to the IONPs’ polymeric coating, releasing them in weakly acidic conditions [61]. Effectiveness compared to free drug delivery was improved both in vitro and in vivo. Conversely, several IONPs approved for clinical use have been discontinued without scientific explanation, raising concerns about long-term clinical applicability. Cytotoxicity concerns, nonspecific cellular uptake, improved targeting efficiency, and improved contrast in MRI signify that IONP research requires many more breakthroughs before large leaps in clinical applications occur [62]. Despite this, their impressive potential as multifunctional NPs in cancer theranostics will continue to drive research. Besides the three major NPs: silica, gold, and iron oxide NPs, titania NPs can be used for imaging as well, though their development as diagnostics may not be as mature since titania NPs were primarily used as catalysts before. For example, gadolinium-doped mesoporous titania nanobeads have been shown to be beneficial for cancer diagnosis for their photoluminescence and enhanced spin relaxation [63]; titania NP-functionalized In2O3 has found applications in diabetes diagnosis [64].

4.1.3 Quantum Dots QDs are fluorescent semiconductor nanocrystals ranging in size from 4 to 12 nm [65,66]. They have potential as imaging agents in biological applications such as disease detection, assays for drug discovery, single protein tracking, and intracellular reporting. First used as biological probes in 1998 [67], their novelties include: tunable light-emission spectra, greater signal brightness, photobleaching resistance, and the ability to produce multiple fluorescent colors at once due to a broad absorbance spectra [68]. At the moment, there are QD products for in vitro imaging on the market such as Qdot Nanocrystals, Nanodots, TriLite Nanocrystals, eFluor Nanocrystals, NanoHC etc.; however, they are reserved for research use only [69]. The detrimental effect of QDs to both humans and the environment remains the greatest obstacle to clinical translation [70]. QDs are made up of an inorganic core, inorganic shell, and an aqueous, organic coating onto which targeting biomolecules can be conjugated [65]. Fig. 4 3 presents a schematic illustration of the general structure of a QD-modified form for photodynamic therapy (PDT). The inorganic shell, traditionally made from a material with a wider band gap, such as zinc sulfide, prevents the toxic core from causing biological harm via increased stability and enhances photoluminescence efficiency. The water-soluble, nonpolar, organic coating forms colloidal dispersions. The core contains Cd21, Se22, or Te22; its size and composition determine the color of the emission and can be tuned during synthesis. Biofunctionalization of QDs uses caps/ ligands to provide three functions: linkage to QD, water solubility, and linkage to targeting biomolecules [71]. Therefore the surface coating of QDs usually requires a bifunctional ligand or amphiphilic molecule, binding to the QD surface with the polar end protruding outwards [72]. Many water solubilization methods exist and are chosen based on application, as biological and physical properties of QDs are greatly influenced by their surface coating.

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FIGURE 4–3 Schematic structural illustration of QDs (A), and summarized mechanism of PDT involving QDs (B). PDT, Photodynamic therapy.

Silica-coated QDs are an alternative also being researched. Bioconjugation with targeting molecules such as peptide antibodies that direct QDs to tumor sites has also been studied. QDs have been used as virus detectors to detect H9 avian influenza virus based on antigen antibody reaction [73]. While metal QDs have overcome many limitations of organic fluorophores, they themselves are not without their own issues. Due to the cytotoxicity of the core metals, such as cadmium, research is also being conducted on other forms of QDs made up of carbon or silicon. Nanomaterial-based diagnostics have opened new frontiers for ultrasensitive detection of bacteria, DNA, and cancer that were inconceivable before. In the future, diagnostic nanodevices could even be implantable in cancer patients for remote live cancer monitoring. Although this 24-h surveillance does raise ethical concerns, it is reasonable to believe nanodiagnostics will potentially play a major role in the development of personalized medicine in the next decade [74]. We envision nanomaterial-based diagnostics will continue making a great impact on the current imaging techniques in the upcoming years.

4.2 Therapeutics According to the World Health Organization, cancer is expected to account for 12 million deaths in 2030 [75], that is approximately 1.5 times the population of New York [76]. Traditional medicine suffers from many drawbacks, such as low selectivity, short circulation,

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FIGURE 4–4 Historical timeline of therapeutic nanosystems from the 1960s to the present day.

and causes chemoresistance, just to name a few; this has propelled researchers to develop nanomedicines. Therapeutic nanomaterials can be roughly divided into two groups, hard and soft. Hard nanomaterials generally refer to metallic, ceramic, and oxide NPs, while soft nanomaterials are primarily comprised of liposomes and polymerics. Fig. 4 4 presents a historical timeline of therapeutic nanosystems. The therapeutic capabilities of inorganic NPs are well established and reviewed in many literatures [37,77,78]. This section of therapeutic nanomaterials will focus on the clinical translations of polymeric nanomaterials and lipidbased nanomaterials.

4.2.1 Polymerics The use of natural and synthetic polymers for medical applications in imaging, diagnostics, and drug delivery is an important and growing section of nanomedicine with many polymer nanodrugs already approved by the FDA and even more in clinical trials [79]. They are perhaps the most easily synthesized and widely applicable entity in the entire field as polymeric NPs can span the entire nanoscale from a single polymer chain to large aggregates, depending on the therapeutic purpose [80]. Essentially, polymer nanodrugs can be divided into two categories: (1) polymer drug conjugates that increase solubility, biocompatibility and increase drug half-life and circulation time in vivo and (2) slowly degrading polymer forms for controlled-release applications. This is the basic, first-generation polymeric NP first designed in the 1960s [55]. To improve efficiency and control of drug release, the properties of the polymeric nanomaterials can be stimulated by a change in their environment (i.e., pH, temperature, UV exposure). The next step, the third generation, has a plurality of functionalities, allowing for controlled targeting and release of hydrophilic and hydrophobic drugs.

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In this section we discuss recent breakthroughs in polymeric nanomaterials, some common polymer drug conjugates used today and other common polymer-based bio-nanotherapeutics such as polymeric micelles, dendrimers, and protein drug and protein polymer drug conjugates. One of the major challenges of translating targeted drug-delivery systems from the benchtop to commercial application is the transport of useful therapeutic molecules through the circulatory system and bloodstream without being attacked by the reticuloendothelial system before it is able to reach its destination. In 1994, Langer et al. [81] published a groundbreaking paper in which PEG was grafted to polymeric NPs as a protective exterior to extend blood circulation time [82]. Also known as polyethylene oxide, PEG polymer has long been a successful synthetic material for surface modification of drug carriers [83]. Composed of repeating ethylene oxide monomeric subunits [84], PEG is soluble in both organic and hydrophilic solvents and is commonly conjugated to other polymers to reduce their hydrophobicity and prevent immune recognition, thus extending the half-life of the molecule in vivo. This occurs because of the hydration layer and steric barrier surrounding the polymeric core, preventing proteins in blood from binding nonspecifically to the particle, limiting its removal from the bloodstream. Recently, ADYNOVATE, a PEGylated antihemophilic factor VIII was approved for the treatment of hemophilia A [79]. PEG increases the half-life of the drug in comparison to non-PEGylated factor VIII, therefore reducing the need for frequent injections. Infrequent injections reduce the risk of antifactor VIII antibody generation leading to a reduced risk for drug inefficacy over time, which occurs in 30% of patients [85]. Another drug recently approved, PLEGRIDY, a PEGylated interferon gamma beta-la protein for treatment of relapsing multiple sclerosis extended daily administration to every 2 4 weeks due to improved biological half-life. PEG is the most well-established polymer and gold standard for polymer NPs. Studies have shown PEG’s permanence properties are influenced by polymer length and surface density on the surface of NPs [86,87]. Longer, less densely packed PEG favors a mushroom conformation, whereas shorter, densely packed PEG prefers a brush-like conformation. A higher surface coverage commonly increases the duration of NP circulation and the ideal surface coverage for maximal NP half-life requires a PEG molecule with a molecular weight of approximately 5000 Da. However, the use of PEG is not a perfect method of camouflage for intravenously injected polymer NPs. Accelerated blood clearance of PEGylated NPs from production of antibodies due to repeated administration has been proposed, thus indicating a reduced drug efficacy over time [88,89]. While optimizing the length and density of surface-coated PEG can suppress an immune response, further research is also being conducted to develop other novel NPs with better compatibility and improved performance. In contrast to PEG, poly[N-(2-hydroxypropyl)methacrylamide] (HPMA) is another hydrophilic polymer that has gained much interest in nanotherapeutics in recent years due to its hydrophilicity, biocompatibility, and lack of immunogenicity [90]. HPMA has multiple functionalization sites. This allows for multiple drugs to be conjugated covalently and codelivered. Polycation DNA micellar complexes have shown improved stabilization when grafted with HPMA compared to PEG [91]. HPMA shells surround a biodegradable polymeric core

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or gene-delivery vehicle, preventing recognition by plasma proteins. HPMA copolymers are commonly used as a conjugated-drug carrier for cancer therapy due to their enhanced permeability and retention in the bloodstream. The first HPMA copolymer conjugate entered clinical trial as an anticancer polymer in 1994, followed by many others including FCE28068, PNU166945, MAG-CPT, and AP5280 [92]. Unfortunately, none has yet been adopted for clinical route use. HPMA polymers have poor cellular internalization due to poor cell membrane affinity. Recently, a folate-decorated, charge-switchable doxorubicin-loaded HPMA conjugate copolymer showed high selectivity and effectivity in kill tumor cells offering a new approach to cancer therapy [93]. HPMA is also frequently grafted with other polymer monomer units to create copolymers that combine useful individual properties of both polymers to improve overall functionality of the nanocarrier. Many of these copolymers self-assemble based on their hydrophilic and lipophilic properties, forming polymeric micelles. Polymeric micelles are round NPs with an inner hydrophobic core and an outer hydrophilic shell made up of amphiphilic copolymers. Loading the inner core with hydrophobic drugs and slowly releasing them over time makes micelles an attractive drug-delivery vehicle [94]. The size, drug loading, and release parameters of the micelle can be controlled by modifying the hydrophilic/hydrophobic balance of the NP. Polymeric micelles have shown great promise in reducing side effects and decreasing toxicity of cancer drugs. Presently, Estrasorb is the only FDA-approved micelle, a transdermal treatment for vasomotor symptoms of menopause. But there are several others in late stages of clinical trials. An example is BIND-014, a micellar prostate cancer NP consisting of the cancer drug Docetaxel in its hydrophobic core conjugated with a ligand that recognizes prostate-specific membrane antigen surrounded by a hydrophilic PEG shell [95]. Polymer micelles have also been demonstrated to be susceptible and thus controlled through changes in their environment [96,97]. A recent example is a reverse polymeric micelle made of PEG-b-PLGA, PEG-phospholipid, and PLGA as a pH-sensitive protein carrier, whose structure is shown in Fig. 4 5 [98]. Protein is only released from the hydrophilic core at neutral or basic pH, potentially useful as an oral drug-delivery carrier.

FIGURE 4–5 Structure of a reverse polymeric micelle made of PEG-b-PLGA (A). (I) SEM and (II) TEM images of reverse polymer micelles (B). TF-1 cell proliferation activity of EPO released from the reverse polymer micelles (C).

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Dendrimers, “dendron” translated from “tree” in Greek, are monodisperse polymers grown in a fractal manner and branch out from a central point [99]. They are spherical, symmetric, repetitively branched polymers. Since their first discovery by Fritz Vogtle in 1978, dendrimers have evolved rapidly in terms of their structural complexity as well as potential applications [100]. In the field of cancer therapy alone, a broad spectrum of subjects has been extensively researched, ranging from dendrimer-based prodrugs, delivery systems to anticancer vaccines [101]. Traditionally, dendrimers are generally synthesized either convergently or divergently; the convergent approach initiates the process from the dendrimer periphery then proceeds inwardly toward the core by repeating the coupling and activating steps, while the divergent approach operates in the exact opposite direction [102]. There are two synthetically unique advantages of dendrimer drug carriers: one is their generation-based size and the other is their monomolecular-nature-allowed formulation stability [101]. In other words, the dendrimer size can be tailored to fit application specificity and because a dendrimer is essentially one macromolecule in unity, thus its synthesis is more reproducible. However, dendrimer synthesis generally involves tedious repetitions of protection, activation, and purification. Thus more efficient synthesis plans have been devised, such as branched monomer addition, orthogonal coupling growth, and metal catalyzed cycloaddition [103]. Furthermore, to explore structural diversity such as internal functional group or multiple peripheral groups, novel schemes such as Passerini reaction and olefin cross-metathesis [104]. Surface modification of dendrimers is useful to increase plasma half-life, increasing permeability, and reducing dosage quantity. Additionally, dendrimers can be easily functionalized with small functional groups, ligands, or PEG, making them ideal candidates for targeted, specific drug delivery [105]. Poly(amidoamine) dendrimers (PAMAM) are the most investigated for drug delivery [106]. With terminal carboxylic, hydroxyl, and amine functional groups, as well as being water-soluble, biocompatible and nonimmunogenic, PAMAM dendrimers contain tertiary amines and amide linkages. These polymeric arms are pH-sensitive, encapsulating or releasing drugs in its cavities depending on the pH of its environment. The different terminal groups also allow both positive and negative charges, so increasing versatility. PAMAM dendrimers have been shown to be cytotoxic and more in vivo studies should be performed before they are considered viable pharmaceutical nanocarriers. An alternative to synthetic polymers, proteins are naturally occurring biocompatible and biodegradable molecules with unique functionality, strong nonantigenicity, renewability, and many potential applications in nanomedicine [107,108]. Their amphiphilic nature allows for interaction with both hydrophilic and hydrophobic phenomena and they have many surface modification sights for drug or ligand conjugation to increase NP specificity. One of the first FDA-approved protein NPs was Abraxane, in 2005. Abraxane consists of the cancer drug Paclitaxel bound to the serum protein albumin. Conjugation with human serum albumin reduces toxicity, increases passive accumulation in tumors, and eradicates the use of the toxic solvent Cremophor, previously needed for paclitaxel delivery, as well as antihistamines and dexamethasone required to prevent an immune reaction [109]. This interest in albumin as a drug carrier has sparked interest in other albumin NPs currently in clinical trials.

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Conjugation of proteins and polymers has improved protein half-life in the delivery of enzymes to combat cancer and other diseases. PEGylation of proteins increases stability, solubility, and reduces protein immunogenicity [110,111]. Gelatin, a denatured protein obtained from collagen, has also been studied extensively as a drug carrier protein. PEGylation of gelatin prolongs circulation time and sterically repulses other proteins binding to the carrier, preventing opsonization [112]. PEGylated-thiolated gelatin NPs for DNA delivery were recently developed [113]. Surface modification of PEG prevents uptake by the reticuloendothelial system, enhancing stability of encapsulated plasmid DNA, and demonstrating potential as a gene therapy vector. Recent research has incorporated gelatin with other biomaterials to form composites which will be discussed in a later section. Other naturally occurring proteins and polymers commonly used in drug-delivery research are starch, dextran, pullulan, pectin, alginate, chitosan, hyaluronic acid, and collagen.

4.2.2 Liposomes A liposome refers to an artificial vesicle of spherical shape with at least one phospholipid bilayer. The genesis of liposome can be traced back to Saunders et al., who discovered in the late 1950s that simple hydration of dry lipid films could produce spherical vesicles; and decades of exploration of lipid-based drug-delivery systems thus began [114]. The first commercialized liposome-based drug, Ambisome for the treatment of visceral leishmaniasis, entered the market in 1997 [115]. Since then, liposomes have attracted much research attention due to their remarkable versatility. Recently, researchers patented a liposome-enabled tattoo removal technology; the liposomes were designed to target the ink pigment and eventually get eliminated by macrophages or transferred to lymph nodes, which significantly improved the tattoo fading speed, offering a painless laser-free alternative [116]. In another report, liposomes were trialed as “heat-triggered grenades” to tackle cancer; the liposomes were capable of controlled abrupt release by taking advantage of the cancer/tumor temperature difference, leaving healthy cells unharmed [117]. From tattoo removal to cancer therapy, the potential applications of liposomes are vast and diverse. The advantages of using liposomes as drugdelivery carriers include but are not limited to: 1. Codelivery function, carrying hydrophilic drugs inside the central cavity and hydrophobic drugs within the phospholipid bilayer, without compromising the encapsulation consistency and release stability; 2. Edge-activator-induced deformability, increasing release persistency and drug retention; 3. Functionalizable surface for targeted release and enzymatic degradation prevention, thus improving efficacy. In the following section of the review we will delve deeper into the codelivery, deformability, surface modification, and controlled-release aspects of modern liposomes from the past decade, with focuses on their formation methods, general performances, and clinical translation implications.

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The underlying principle of liposome formation is self-assembly, an enthalpy-driven process in which components, such as lipids, edge activators, and drugs of various natures, rearrange themselves into a discernible structure exploitable for applications. At the laboratory scale, the most commonly used methods are modified ethanol injection [118], reverse phase evaporation [119], and thin-film hydration [120]. However, these conventional methods are often plagued by lack of total control over parameters such as size, charge, polydispersity, and lamellarity. As a result, the value of liposomes as drug carriers is diminished by prevalent batch-to-batch inconsistency. Furthermore, industrial production scale-up and point-ofcare scale-down only exacerbate the problem, making conventional methods extremely difficult to implement. For example, industrially, the homogenization of liposome size and lamellarity is usually achieved by a very high-pressure pump through continuous colliding [121]. In attempts to overcome the bottleneck of conventional methods, a number of modified and novel formation methods have been devised, aiming to improve and optimize liposome fabrication in the past decade. Santo et al. proposed a new supercritical CO2-assisted continuous process for liposome formation in which water in CO2 emulsion was formed. The mechanism was attributed to favorable interactions between expanded liquid phospholipid and atomized water droplet; and the resulting product was reported to be uniform with high encapsulation efficiencies of 85% 90% [122]. Elersˇicˇ et al. demonstrated a modified electro-formation method for magnetic targeted drug-delivery liposome formation and discussed size dependence on amplitude, as well as frequency of the applied electric field [123]. Kastner et al. designed a microfluidic-controlled method for liposome formation in which precise control over the mixing rate and ratio between aqueous and solvent streams was possible. Incorporating a chaotic advection micromixer and a staggered herringbone micromixer into the production setup, the process showed remarkable reproducibility and scalability in terms of size control, loading efficiency, and overall throughput for liposomes ranging from 50 to 450 nm [124]. Thus the microfluidic method has been proven not only feasible but also efficient to reduce large vesicles into unilamellar liposomes, replacing primitive mechanical approaches as the new promising candidate for industrial scale-up productions. Supercritical, microfluidic, and electro-formation are only three of many novel methods that are being explored and developed to address the aforementioned issues existing in conventional approaches; with a continued influx of research funding and effort, it is reasonable to expect a new generation of liposome-based drugs entering the mass production pipeline in the near future. Liposomes have two compartments for drug storage purposes; one is within the central cavity formed by surrounding hydrophilic heads, the other is between the hydrophobic tails of the phospholipid bilayer. This unique structural property can be exploited for a codelivery-induced synergetic effect in therapeutics; the space within the cavity and between the bilayer can simultaneously load drugs of various natures, water-soluble and lipidsoluble, respectively. In the past decade, the codelivery property of liposomes has been extensively researched and remarkable progress has been made in many major issues awaiting resolution; one hot topic being multidrug resistance commonly experienced in cancer treatment. Despite its great efficacy in minimizing the side effects of breast cancer drugs,

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doxorubicin hydrochloride (DOX) has been shown to induce multidrug resistance. DNA nanostructures, on the other hand, have been reported to inhibit the unwanted resistance caused by DOX. Therefore giant liposomes, along with porous silicon NPs, were selected to codeliver drugs (Erlotinib and 17-AAG), DOX and DNA nanostructures simultaneously to test against breast cancer MCF-7 cells. The combination showed excellent encapsulation efficiency, loading ability, release stability, and synergism, proving itself a strong candidate for the state-of-the-art drug-delivery carrier for cancer treatment [125]. Other similar reports include the codelivery of epirubicin and lonidamine to circumvent multidrug resistance in lung cancer by altering mitochondrial signaling pathways to trigger cancer cell apoptosis [126], curcumin and albumin-paclitaxel hybrid NP in skin cancer therapy [127], and doxorubicin and SATB1 siRNA for gastric cancer cell growth inhibition [128]. The liposome shell is largely comprised of phospholipid bilayers whose rigidity can be adjusted by using an edge activator to impart liposome-tailored flexibility suitable for specific applications. Indeed, deformable liposomes have experienced rapid development in the past decade, particularly in the field of transdermal drug delivery. The working principle can be briefly explained as a destabilization process whereby introducing a calculated amount of foreign edge activator into the bilayer space helps loosen the tight grip between hydrocarbon tails. Thus it weakens the intermolecular interaction, rendering the overall liposome deformable. Confocal microscopy revealed that intact liposomes tend to remain confined to the upper layers of the stratum corneum [129]. In comparison, deformable liposomes can more effectively penetrate the absorption barrier and reach deeper into the granular epithelial cells, forming a sustainable reservoir and realizing the full potential of drugs delivered. This is especially beneficial for drugs of high molecular weight that often suffer from poor permeability via the mucosal route using conventional methods [130]. For example, deformable liposomes for transdermal insulin delivery patch have been reported to show 50% efficacy as compared to subcutaneous injection [131]. This noninvasive therapeutic approach has the potential to relieve hundreds of millions of diabetes patients of the pain and inconvenience of needle injection [132]. Beside pharmaceutical purposes, deformable liposomes have also found applications in cosmetic products. Quercetin provides cellular protection against ultraviolet radiation-induced skin damage by scavenging oxygen radicals, inhibiting lipid peroxidation, and chelating with metal ions. In spite of having the highest antioxidant action of all flavonoids, the application of quercetin in sunscreens is hindered by its poor percutaneous permeation and low skin deposition. Attempting to improve its hydrophobicity by adding ethanol as a cosolvent is effective and common, however, it often causes local irritation, and thus it is not completely ideal. Deformable liposomes as transdermal quercetin delivery carriers have been investigated and demonstrated high entrapment efficiency, prolonged release profile, increased cell viability, and reduced reactive oxygen species; it was also observed that deformable liposomes can promptly respond to external stress through energetically favorable shape transformation [129]. Similar research has been conducted on other antioxidants, such as catechin and procyanidin [133,134], both resulted in positive outcomes for deformable liposomes. The most commonly used edge activators include propylene glycol, sodium cholate, Span 20, and Tween 80. It is worth noting that propylene glycol has been

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approved as an inactive ingredient and it is believed to be safe to use in pharmaceutical and cosmetic products by the FDA [130]. Although having a promising reputation as delivery carriers of the future, by the end of the 1970s, the swift pace of liposome development was crippled by poor in vivo circulation period and lack of selectiveness in drug release [135]. The former issue was resolved by PEGylation, while the latter remains an area of extensive research to this day. Conventional liposomes are easily recognized and quickly eliminated by macrophages of the immune system. PEGylation was designed to camouflage the liposome and many, such as Doxil, Caelyx, Myocet, etc., have received a great market response. Similarly TPGS-ylation, essentially PEGylated vitamin E, has also achieved preclinical success [136]. Recently, new directions for stealth liposome coatings have been explored. Oral administration of unprotected peptide biopharmaceuticals often suffers from enzymatic degradation; as a result, little can end up getting into the systemic circulation. Attempts to adopt liposomes as a drug-delivery carrier have been made but the outcome was ineffective because continuous contact was necessary for optimal absorption. To address this requirement, thiolated chitosan was coated onto liposomes to increase its muco-adhesiveness. Also, in order to further enhance the stability of thiomers against oxidation by the hostile gastric environment, an extra step of free thiol immobilization for protective disulfide bond formation has recently been devised and proven to increase adhesiveness and permeability by as much as 14-fold and 4.2-fold, respectively [137]. Apart from thiomers, other biomaterials have also been developed as liposome coatings for performance enhancement; for example, oligomannose-coated liposomes have proven effective in delivering antigen and RNA adjuvant to invoke immune responses for the treatment of human parainfluenza viruses as a nasal vaccine [138]. Besides the coating, the liposome surface can also be modified by incorporating polymers, peptides, and antibodies for targeted drug delivery, as shown in Fig. 4 6. As mentioned previously, selectiveness in drug release, especially in cancer treatment, is still an active area of research and targeting can be roughly divided into ligand-based and nonligand-based. It is worth noting that besides the codelivery capability of liposomes explored in preceding sections, another reason behind many recent liposome-enabled pivotal breakthroughs in cancer therapy is due to their preferential accumulation at tumor sites, a phenomenon commonly referred to as the enhanced permeability and retention effect. It is well established that tumors in pathological conditions are surrounded by abnormally constructed angiogenic blood vessels; their large vascular fenestrae and compromised lymphatic drainage grant liposomes access into the interstitium, a region otherwise unreachable by the liposome if cells are healthy [139]. In the category of nonligand-based targeting, the general philosophy is to have loaded liposomes triggered by the surrounding intracellular environment. One approach to triggered release is by utilizing thermosensitive liposomes, an idea first conceived by Yatvin et al., and it has demonstrated numerous advantages over traditional chemotherapy [140]. By exploiting the transitional temperature of surface modifier, equipment such as high-intensity focused ultrasound can induce local hyperthermia, causing liposome leakage and thus abrupt release. Unfortunately, many polymer-based liposome complexes are not biodegradable and

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FIGURE 4–6 Common surface modification for stealth liposomes. (A) Conventional liposome, (B) PEGylated liposome, (C) ligand-targeted liposome, and (D) theranostic liposome.

exhibit toxicity. To tackle this problem, Park et al. employed an elastin-like polypeptide with adjustable transitional temperature to decorate the liposome surface. The novel temperature-triggered liposome showed high loading efficiency, low cytotoxicity, abrupt release at 42 C, but otherwise stable with prolonged half-life at physiological temperatures [141]. ThermoDox is a thermosensitive liposome developed by Celsion Corporation and has entered phase III clinical trial [69]. Another approach to triggered release is by utilizing pHsensitive liposomes. For instance, a pH-sensitive liposome modified by stearoyl-PEG-poly (methacryloyl sulfadimethoxine) was developed by Vila-Caballer et al. as an alternative to transurethral resection widely practiced in bladder cancer treatment. Upon exposure to acidic pH like urine, the liposomes underwent rapid aggregation yielding epithelium adhesion; this behavior provides a selective response to environments, such as solid tumors or the bladder cavity [142]. In the category of ligand-based targeting, the general philosophy is to functionalize liposomes by chemical conjugation for high responsiveness and enhanced uptake. Briefly, the mechanism is based on the coupling of an targeting ligand, such as antibody, protein, peptide, vitamin, carbohydrate, to an overexpressed receptor at the tumor cell surface. Incorporating receptor-mediated endocytosis provides an extra level of sophistication to the passive permeability and retention effect intrinsic to liposomes. A list of ideal target pairings can be found in a review published by Noble et al. [139]. Besides holding great promise in

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cancer treatment, functionalization of liposomes has also found potential applications in treating other diseases. For example, liposomes bifunctionalized with phosphatidic acid and Apo-E-derived peptide were designed for Alzheimer’s disease treatment. The derived peptide was selected to bind and cross the blood brain-barrier, while phosphatidic acid was selected to bind beta-amyloid, whose accumulation is a histological hallmark of the disease. Together, the bifunctionalized liposome was able to achieve 70% inhibition after 72 h and trigger 60% disaggregation in 120 h; the disaggregation was attributed to a synergic action unique to the combination of phosphatidic acid and Apo-E-derived peptide, proving the bifunctionalized liposomes as a valuable nano-device for Alzheimer’s disease therapy [143]. To the best of our knowledge, three ligand-mediated liposomal targeting systems developed for cancer therapy have progressed to clinical trials: the first being MCC-465 with immune shielding PEG decoration and antigen-binding fragment dimers, followed by the similar SGT53-01 and MBP-426 [144]. Codelivery, deformability, and functionalization are only three of the many advantages that liposomes possess for therapeutic applications, and they all have been harnessed skillfully and with great imagination. Although the working principle of liposomes is simple, the real challenge for state-of-the-art liposomes lies. The real challenge for state-of-the-art liposomes lies in the question of how to skillfully combine all of their unique properties to tackle the real-life problems encountered in clinical therapy. The future of nanotechnology in medicine lies in the seamless integration of both diagnostic and therapeutic nanomaterials—namely theranostic NPs, an idea which was only briefly touched upon in previous sections. In fact, there already seems to be a bottleneck for the clinical translation of soft therapeutic nanomaterials, as, since the first targeted liposome was described somewhat 40 years ago, only a handful of systems have ever made it to clinical trials [145]. Incorporating diagnostic functionalities can potentially offer researchers a deep insight into the events taking place at the nano bio interface, as well as gaining new knowledge of the fate of nanosystems at cellular levels [144]. The circumstance at hand pushes for the development of theranostic systems and there have been reviews of theranostic NPs available since early 2010, in which the exciting idea of personalized medicine and potential solutions for obstacles faced are discussed in great detail [146 148]. The current section on therapeutic nanomaterials and the previous section on diagnostic nanomaterials aim to equip readers with the knowledge necessary to gain a deeper understanding of theranostic designs.

4.3 Tissue Engineering Tissue engineering refers to the interdisciplinary field that seeks to create, repair, or replace tissues and organs by using combinations of cells, biomaterials, and biologically active molecules [149]. The definition of tissue engineering was first developed at a scientific meeting sponsored by the National Science Foundation at Lake Tahoe, California, as follows [150]:

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“Tissue engineering” is the application of the principles and methods of engineering and the life sciences toward the fundamental understanding of structure/function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve functions. Reconstruction of cranial defects using gold dates back to 2000 BC and modern tissue engineering emerged in the early 1990s as an alternative to the then-prevalent grafting and alloplastic approaches [151]. From 3D-printed hydroxyapatite scaffold for bone regeneration to self-assembling peptide hydrogel for brain regeneration, the level of sophistication that tissue engineering has reached in the past decade is truly remarkable and may even seem to be straight out of sci-fi movies to nonexperts [152]. It is thus the aim of this chapter to cover the clinical translation of tissue engineering that has taken place recently, with focuses on scaffold and hydrogel.

4.3.1 Scaffolds Scaffolds have attracted much research interest in tissue engineering due to their structural similarity to the natural extracellular matrix, which provides an ideal biomimetic environment for tissue regeneration by promoting initial cell attachment and subsequent tissue regeneration. Scaffolds are favored over traditional surgical implantation procedures for their noninvasive nature, low infection rate, and multifunctionality, such as controlled release, environmental responsiveness, tuned biodegradability, etc. The ultimate goal for a scaffold is to act as a temporary surrogate until the damaged or lost functions in the host tissue are repaired or restored. Ideally, the scaffold should be degraded and absorbed at approximately the same rate as the native extracellular matrix regeneration in this gradual process. At present, scaffolds have found a variety of applications in the fields of orthopedic, craniofacial, and dental surgeries (Fig. 4 7). It has been reported that the market for smart biomaterials such as the state-of-the-art scaffold is expected to reach $113 billion by 2025 [153]. In the following section, we will first briefly discuss the scaffold material and fabrication method, then move on to examining how smart scaffold regulates the tissue engineering process in its recent research development and future clinical translations. Scaffold can be fabricated or biotemplated from a wide spectrum of materials. Natural coral exoskeleton for scaffold fabrication has been adopted for bone defect regeneration since the early 1970s. The histoarchitecture of coral is an interconnected porous structure, ideal for nutrient and waste transport, and has demonstrated numerous advantages such as biocompatibility, biodegradability, bioavailability, as well as osteoconductivity [154]. Natural polymers such as collagen, agarose, gelatin, and chitosan are also appealing options. It is worth noting that collagen has been adopted as the clinical standard for the delivery of rhBMPs, a human bone morphogenetic protein, in systems such as Medtronic INFUSE and Olympus OP-1 [155]. In the meantime, synthetic polymers such as polyester, polyanhydride, polyphosphazene, and polyurethane are gaining popularity as scaffold materials for their controlled structure, high processing flexibility, and low immunological concerns [156].

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FIGURE 4–7 The concept of skeletal tissue regeneration via scaffold-based tissue engineering strategies (A), and representative internal structures of porous scaffolds (B) produced via (I) polymer sponge replication, (II) impregnate sintering, (III) gelcast foaming, (IV) solid free-form fabrication, (V) solvent casting and particulate leaching, (VI) phase separation, (VII) microsphere sintering, and (VIII) electrospinning.

Polymers from the polyester family, such as polyglycolic acid and polylactic acid (PLA), are particularly favored for their biodegradability. Ceramic materials, especially hydroxyapatites, are ideal scaffold materials, too. Hydroxyapatite is the main inorganic salt of human bone. Sharing chemical similarity with the mineralized phase of biologic bone, hydroxyapatite is osteophilic, osteoconductive, and osteointegrated; hydroxyapatite’s ability to form a direct chemical bond with human bone has been reported and accounts for its excellent osteogenesis property [157]. Many hydroxyapatite-based nanomaterials have been approved by the FDA as bone substitutes in the past decade, such as Ostim, OsSatura, NanOss, EquivaBone, CarriGen, Alpha-bsm, Beta-bsm, and Gamma-bsm [69]. It is common to see multiple materials combined together forming hybrid scaffold to compensate for each individual application-specific drawback or disadvantage for further overall performance enhancement. For example, Kim et al. demonstrated a hydroxyapatite/alginate/chitosan scaffold in which hydroxyapatite and alginate were incorporated as mechanical and solubility reinforcements,

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respectively. The composite was reported to have uniform pore structure and thus increased mechanical properties such as compressive strength and elastic modulus [158]. The macro- and microstructural properties of scaffolds have been shown to not only affect cell survival, signaling, growth, propagation, and reorganization, but also play major roles in influencing cell shape and gene expressions that relate to the preservation of native phenotypes. For example, scaffolds with pore size between 20 and 125 μm are a prerequisite for regenerating adult mammalian skin, while sizes below 200 μm are rarely used for orthopedic applications [159]. Therefore when it comes to the clinical translation of scaffolds, the best course of action for fabrication method selection is to examine the application requirement first and then choose accordingly. Conventional techniques for scaffold fabrication include, but are not limited to, solvent casting, particulate leaching, membrane lamination, and melting molding [160]. However, conventional methods often suffer from a few drawbacks that limit their clinical translation: conventional methods require multiple processing steps and are time-consuming; structural interconnectivity and precise balance of the trade-off between mechanical robustness and optimal porosity are not entirely reproducible, making it difficult for industrial scale-up; certain conventional methods, such as electrospinning, involve extensive use of toxic organic solvent that, if not properly eliminated, can lead to detrimental health effects; conventional methods cannot meet the demand for customizable scaffolds with complex architectures. Computational topology design enabled solid free-form technologies, including selective laser sintering, fused deposition modeling, electron beam melting, and three-dimensional (3D) printing, to be presented as alternatives to overcome the current limitations in conventional techniques [161]. Besides providing mechanical and biological support as a native extracellular matrixresembling framework, tissue engineering scaffolds have also been developed as substrates to carry out active therapeutic tasks. Scaffolds provide an excellent platform for loading various drugs and administering sustained release as a stationary carrier in vivo. Antimicrobial agents such as silver, gentamicin, and doxycycline can be encapsulated within a degradable matrix to combat implant-associated bacterial infection, remove existing pathogens, and allow bone repair to occur in sterilized conditions for up to months; antibiotic-releasing scaffolds have been adopted clinically in orthopedics for a number of years [162]. Multifactorial and sequential release has also been developed in tissue engineering scaffolds for synergetic effects as well as to meet application-specific requirements, for example: repairing injured spinal cord necessitates the incorporation and codelivery of multiple neurotrophic factors [163]; the order of triggering stem and progenitor cells migration to injury sites first, then activating osteoblastic differentiation next is a well-observed phenomenon for optimal bone regeneration in orthopedics [164]. Shape-memory scaffold capable with high compressibility designed to minimize surgical invasiveness and reduce scar formation is another current active area of research [165]. Several scaffolds that have entered clinical trials in the past decades include the Neuro-Spinal Scaffold developed by InVivoTherapeutics to facilitate new neuronal connections in spinal cord injury and L C Ligament by Humacyte to facilitate regrowth of the anterior cruciate ligament in knees [166].

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4.3.2 Hydrogels First described in 1960 by Wichterle and Lim [167] for potential use as a contact lens, hydrogel research has expanded vastly in the field of biomaterials in recent decades. Hydrogels are hydrophilic, crosslinked, 3D polymeric networks that swell with water/biological fluid but do not dissolve in aqueous solution [168]. They can be fabricated from natural/synthetic polymers or a combination of the two and are either physically or chemically crosslinked. Due to their biocompatibility and controllable porosity they have potential in drug delivery and due to their biodegradability, softness, and high water content they are useful in the field of tissue engineering. These properties make hydrogels the synthetic biomaterial most alike to naturally living tissue [169]. The water-swelling capacity of the polymer chains is tunable, ranging from 10% to 20% to many thousands of times their dry weight in water [170]. Hydrogels can be called “physical” or “reversible” if ionic bonds, hydrogen bonds, hydrophobic forces, or molecular entanglements are used to hold them together. Likewise, hydrogels are called “chemical” or “permanent” if polymer chains are covalently crosslinked. Some natural polymers used in hydrogel fabrication include: hyaluronic acid, alginate, chitosan, collagen, gelatin, fibrin, and agarose. Some synthetic polymers used in hydrogel fabrication include: PEG; PLA; PLGA, poly(lactic-co-glycolic acid); PCL, polycaprolactone; PAAc, poly(acrylic acid); PVA, poly(vinyl alcohol); PBR, poly[(r)-3-hydroxybutyrate]; PNIPAAm, poly(N-isopropylamide); and polypeptides. These are just a few of the many polymers being tested in research labs, in preclinical and clinical trials. This section will further discuss different methods of fabrication and various applications, such as contact lenses, hygiene products, wound dressing, drug-delivery vehicles, and tissue engineering matrices. There are many different hydrogel fabrication methods, dependent on the physical or chemical crosslinking of the hydrogel. Physically crosslinked hydrogels are comparatively weaker but do not require crosslinking agents. “Sol gel” chemistry or reverse thermal gelation can be used to crosslink hydrophobic polymer domains of amphoteric polymers, otherwise known as “gelators” [171]. Soluble at low temperatures, upon heating hydrophobic domains aggregate, minimizing the surface area exposed to water. The gelation temperature is dependent on polymer concentration and chemical structure (size of hydrophobic segment, molecular weight). A gelation temperature close to physiological temperature is potentially useful as a thermosensitive hydrogel is a liquid at room temperature but gelates in situ. The larger the hydrophobic segment, the stronger the hydrophobic aggregation, the lower the gelation temperature. Ionic interactions, either between a charged polymer and a charged molecule or between two oppositely charged polymers, has also been used to create hydrogels. These hydrogels are advantageous because other ionic molecules inside the extracellular matrix compete with gel components, slowly causing degradation of the matrix over time. Furthermore, ionic functional groups responsible for gelation can be protonated or deprotonated via pH changes, thus controlling overall gelation. Hydrogen bonding interactions can also produce hydrogels, such as the in vitro procedure for freeze thawing PVA. Additionally, synergistic effects can occur between two polymers due to compatible geometries. These hydrogen bonds are not strong enough to withstand shear force and can thus be

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injected through a syringe. Hydrogels forming from stereocomplexation arise from synergistic interactions between polymers of the same chemical composition but different stereochemistry. An example of this is the stereochemistry of L and D polylactide blocks. These hydrogels are limited by their composition as the inclusion of other polymers can alter the gel entirely. In recent years, supramolecular chemistry has also been used to form hydrogels, often using cyclodextrins and poly(alkylene oxide) polymers [172]. Geometric compatibilities between hydrophobic polymer domains and cyclodextrin centers allows threading and placement of polymer chains inside them. This property can be combined with other interactions to produce stronger gels such as the work done by Li et al. [173], which combines hydrophobic interactions of the PHB in PEG PHB PEG triblock copolymers with supramolecular chemistry between PEG and alpha-cyclodextrin. Chemically crosslinked hydrogels are formed by covalent bonds, are generally stronger, degrade at a slower rate, and have better design flexibility. One common method of covalent crosslinking, free radical polymerization, uses a crosslinking agent and initiator molecule to polymerize hydrophilic monomers [174]. The initiator is usually a free-radical-generating molecule that reacts with vinyl monomers to create elongating chains, randomly crosslinked by the crosslinking agent [175]. Excess initiator and monomers may not be biocompatible and thus be removed via purification, a time-consuming process. Radical polymerization also forms hydrogels by crosslinking water-soluble polymers. Methacrylic groups have been conjugated to hydrophilic polymers and crosslinked together with an initiator. Polymers containing functional groups (COOH, NH2, OH) can undergo addition or condensation reactions with crosslinker molecules to form covalent bonds between polymer chains [176]. Reactions such as amine-carboxylic acid, isocyanate-OH/NH2, and Schiff base formation have been used extensively in hydrogel research. UV, gamma, electron beam radiation, etc. can also induce radicals due to high energy, creating a linked mesh upon exposure (Fig. 4 8). Hydrogels provide an intriguing drug- and cell-delivery vehicle because of their variable porosity, biodegradability, and similarity to the extracellular matrix providing a controllable, nontoxic environment for targeted delivery. Furthermore, loaded cargo is protected from degradation by enzymes or hydrolyzation [177]. For example, injectable microsphere hydrogel has been shown to be excellent for stem bone regeneration required cell transplantation due to its minimal invasiveness, enhanced engraftment, and in vivo stability. GelMA microspheres were fabricated via a microfluidic method and bone marrow-derived mesenchymal stem cells were embedded; they were then subject to osteogenesis tests in vitro and in vivo [178] (Fig. 4 9). Mechanisms for controlled release of drugs from hydrogel: diffusion controlled, swelling controlled, chemically controlled, and environmentally responsive release. A reservoir delivery system is made up of a hydrogel surrounding an enclosed drug core (i.e., capsule, sphere, cylinder, or slab) [179]. Drugs are released via diffusion from the highly concentrated core over time. A second diffusion-controlled method is a matrix system where the drug is dissolved throughout the hydrogel and is initially released proportionally to the square root of time. Swelling controlled systems are similar; a drug is dissolved in a polymer hydrogel that begins to swell and expand upon contact with biofluid allowing the drug to diffuse out.

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FIGURE 4–8 Chemically crosslinked hydrogel synthesis by UV, gamma, or electron beam radiation. (A) shows the cross-linking process from water-soluble polymer to hydrogel. (B) shows three major cross linking categories: physical, chemical and biological crosslinking and provides examples for each, from which tissue engineering scaffolds have derived applications from.

Tissue engineering is the development of engineered materials to create synthetic tissues or organs to replace real tissue. Hydrogels are applicable as a space-filling agent, bioactive substance delivery vehicle, and as a 3-D structure that organizes cells and stimulates tissue growth [174]. There are several physical and biological parameters belonging to hydrogels that must be met in order for a material to be useful in tissue engineering, such as

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FIGURE 4–9 Injectable stem cell-laden microsphere hydrogel for rapid generation of osteogenic tissue constructs. (A) shows the microfluidic fabrication process of injectable microsphere hydrogels and its application to test subjects. (B) shows the live/dead and actin/nuclei test results of injectable microsphere hydrogels.

being mechanically robust, biodegradable, good cell adhesive and above all, biocompatible. 3D bioprinting has become increasingly relevant in tissue engineering in recent years. Hydrogels show promise mimicking tissues’ self-healing ability, its capability to intrinsically repair itself; an exceedingly important property that prolongs tissue life and retains original properties [180]. The paradigm shift from surgical graft substitution procedure to modern utilization of scaffolds and hydrogels is poised to inspire new designs in the field of tissue engineering. Recently, a truly creative idea of combining scaffold and hydrogel was proposed by Cui et al. to address the distal necrosis issue of skin flap commonly encountered in plastic surgery. The hydro-scaffold was fabricated by electrospinning photocrosslinkable GelMA under UV light, as shown in Fig. 4 10. The hydro-scaffold for accelerated vascularization demonstrated tunable mechanical flexibility and adjustable degradation profile [181]. If more practical

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FIGURE 4–10 Schematic illustration of hydrogel scaffold fabricated by electrospinning photocrosslinkable GelMA under UV light (A). The morphology of electrospun fibers of GelMA (B). Vascularization immunofluorescence staining of CD31 (C), and scheme picture (D).

ideas are incorporated from the previous sections of diagnostic and therapeutic nanomaterials, tissue engineering is bound to make greater leaps; the current optimism is well founded that one day a truly smart design will not only provide required support as a placeholder, but also be able to simultaneously live monitor the regeneration process and administer proper doses of multiple drugs autonomously, thus fundamentally transforming the healing experience for patients.

4.4 Conclusion and Perspective Nanomedicine is a burgeoning field of research with limitless possibilities for quality-of-life improvement for patients. With regard to the prospective direction of nanomedicine development, most researchers believe in pushing a landscape of more advanced multicomponent nanomaterials that target different pathologies simultaneously [182]. However, there are some who favor a simpler formulation rationale for smart nanomedicine, which is more likely to become a “translatable and applicable” part of clinical reality [183]. In this review, we have delved into the clinical translation of nanomaterials, including CNTs, INPs, and QDs from the diagnostic section, polymerics and liposomes from the therapeutic section, and scaffolds and hydrogels from the tissue engineering section. Besides the tremendous

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progress illustrated in each individual field above, there are extra criteria to consider if one wishes to accelerate the clinical translation of nanomaterials. First, the actual benefits brought by the nanomedicine have to balance, or ideally exceed, its production cost [36]. Unfortunately, this topic is beyond the scope of this chapter; and to the best of our knowledge, there are very few comprehensive reviews written from the economical perspective of nanomedicines [184]. Second, the effect of physicochemical properties of nanomaterials, as well as the mechanism, on their toxicity remain unclear and must be understood before entering clinical trial [185 189]. A successfully clinical translation of any nanomaterial is never an easy feat; it requires extensive preclinical research, carefully selected clinical indication, proper design, and faithful execution of clinical trials [190]. However, we are optimistic that with unwavering research endeavors and substantial funds, more clinical translation of nanomaterials will be approved and enter the production pipeline in the near future.

Acknowledgment This work was supported by the Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support (20171906).

References [1] Y. Dang, Y. Zhang, L. Fan, H. Chen, M.C. Roco, Trends in worldwide nanotechnology patent applications: 1991 to 2008, J. Nanoparticle Res. 12 (3) (2010) 687 706. [2] H.M.E. Azzazy, M.M.H. Mansour, In vitro diagnostic prospects of nanoparticles, Clin. Chim. Acta 403 (1 2) (2009) 1 8. [3] N.K. Mehra, V. Mishra, N.K. Jain, A review of ligand tethered surface engineered carbon nanotubes, Biomaterials 35 (4) (2014) 1267 1283. [4] Y. Yang, China focus: China aims high in nanotechnology - Xinhua | English.news.cn, XINHUANET. Available: ,http://news.xinhuanet.com/english/2017-08/30/c_136568055.htm., 2017 (accessed 05.02.18). [5] Darren Waters, BBC NEWS | Technology | Nano switch hints at future chips, BBC NEWS. Available ,http://news.bbc.co.uk/2/hi/technology/7352464.stm., 2008 (accessed 05.02.18). [6] J. Gallagher, Heat-triggered ‘grenades’ hit cancer BBC News, BBC NEWS. Available: ,http://www. bbc.com/news/health-34667804., 2015 (accessed 05.02.18). [7] M.P. Birch, J.F. Messenger, A.G. Messenger, Hair density, hair diameter and the prevalence of female pattern hair loss, Br. J. Dermatol. 144 (2) (2001) 297 304. [8] S. Kumar, W. Ahlawat, R. Kumar, N. Dilbaghi, Graphene, carbon nanotubes, zinc oxide and gold as elite nanomaterials for fabrication of biosensors for healthcare, Biosens. Bioelectron. 70 (2015) 498 503. [9] S. Iijima, T. Ichihashi, Single-shell carbon nanotubes of 1-nm diameter, Nature 363 (6430) (1993) 603 605. [10] J. Morgan, First computer made of carbon nanotubes is unveiled BBC News, BBC News. Available: ,http://www.bbc.com/news/science-environment-24232896., 2013 (accessed 05.02.18). [11] T. Doran, K. Scott, Vantablack: darkest material known to man set to shine at Winter Olympics CNN, CNN News. Available: ,https://edition.cnn.com/2017/11/15/world/vantablack-blackest-black-material/ index.html., 2017 (accessed 05.02.18).

Chapter 4 • Clinical Translation of Nanomaterials

103

[12] Z. Liu, J.T. Robinson, S.M. Tabakman, K. Yang, H. Dai, Carbon materials for drug delivery & cancer therapy, Mater. Today 14 (7 8) (2011) 316 323. [13] Y. Zhang, Y. Bai, B. Yan, Functionalized carbon nanotubes for potential medicinal applications, Drug Discov. Today 15 (11 12) (2010) 428 435. [14] F. Liang, B. Chen, A review on biomedical applications of single-walled carbon nanotubes, Curr. Med. Chem. 17 (1) (2010) 10 24. [15] S. Beg, M. Rizwan, A.M. Sheikh, M.S. Hasnain, K. Anwer, K. Kohli, Advancement in carbon nanotubes: basics, biomedical applications and toxicity, J. Pharm. Pharmacol 63 (2) (2011) 141 163. [16] C.D. Fahrenholtz, M. Hadimani, S.B. King, S.V. Torti, R. Singh, Targeting breast cancer with sugarcoated carbon nanotubes, Nanomedicine (Lond). 10 (16) (2015) 2481 2497. [17] A. Bianco, M. Prato, Can carbon nanotubes be considered useful tools for biological applications? Adv. Mater. 15 (20) (2003) 1765 1768. [18] M. Prato, K. Kostarelos, A. Bianco, Functionalized carbon nanotubes in drug design and discovery, Acc. Chem. Res. 41 (1) (2008) 60 68. [19] L. Lacerda, A. Bianco, M. Prato, K. Kostarelos, Carbon nanotubes as nanomedicines: from toxicology to pharmacology, Adv. Drug Deliv. Rev. 58 (14) (2006) 1460 1470. [20] J.-W. Kim, E.I. Galanzha, E.V. Shashkov, H.-M. Moon, V.P. Zharov, Golden carbon nanotubes as multimodal photoacoustic and photothermal high-contrast molecular agents., Nat. Nanotechnol 4 (10) (2009) 688 694. [21] J. Muller, et al., Structural defects play a major role in the acute lung toxicity of multiwall carbon nanotubes: toxicological aspects, Chem. Res. Toxicol. 21 (9) (2008) 1698 1705. [22] D.W. Porter, et al., Mouse pulmonary dose- and time course-responses induced by exposure to multiwalled carbon nanotubes, Toxicology 269 (2 3) (2010) 136 147. [23] Z. Yang, et al., Pharmacological and toxicological target organelles and safe use of single-walled carbon nanotubes as drug carriers in treating Alzheimer disease, Nanomed. Nanotechnol. Biol. Med. 6 (3) (2010) 427 441. [24] S.-T. Yang, et al., Long-term accumulation and low toxicity of single-walled carbon nanotubes in intravenously exposed mice, Toxicol. Lett. 181 (3) (2008) 182 189. [25] C.A. Poland, et al., Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study, Nat. Nanotechnol. 3 (7) (2008) 423 428. [26] Z. Liu, C. Davis, W. Cai, L. He, X. Chen, H. Dai, Circulation and long-term fate of functionalized, biocompatible single-walled carbon nanotubes in mice probed by Raman spectroscopy, Proc. Natl. Acad. Sci. U.S.A. 105 (5) (2008) 1410 1415. [27] S.Y. Hong, et al., Filled and glycosylated carbon nanotubes for in vivo radioemitter localization and imaging, Nat. Mater. 9 (6) (2010) 485 490. [28] R.J. Chen, et al., Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors, Proc. Natl. Acad. Sci. U.S.A. 100 (9) (2003) 4984 4989. [29] S. Ji, et al., Carbon nanotubes in cancer diagnosis and therapy, Biochim. Biophys. Acta 1806 (1) (2010) 29 35.

Rev. Cancer

[30] C. Richard, B.-T. Doan, J.-C. Beloeil, M. Bessodes, É. Tóth, D. Scherman, Noncovalent functionalization of carbon nanotubes with amphiphilic Gd3 1 chelates: toward powerful T1 and T2 MRI contrast agents, Nano Lett. 8 (2008) 232 236. [31] M. Yin, et al., Water-dispersible multiwalled carbon nanotube/iron oxide hybrids as contrast agents for cellular magnetic resonance imaging, Carbon N.Y. 50 (6) (2012) 2162 2170. [32] K. Welsher, S.P. Sherlock, H. Dai, Deep-tissue anatomical imaging of mice using carbon nanotube fluorophores in the second near-infrared window, Proc. Natl. Acad. Sci. U.S.A. 108 (22) (2011) 8943 8948.

104

THERANOSTIC BIONANOMATERIALS

[33] K. Welsher, et al., A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice, Nat. Nanotechnol. 4 (11) (2009) 773 780. [34] A. de la Zerda, et al., Ultrahigh sensitivity carbon nanotube agents for photoacoustic molecular imaging in living mice, Nano Lett. 10 (6) (2010) 2168 2172. [35] B. Chen, et al., Carbon nanotube-based magnetic-fluorescent nanohybrids as highly efficient contrast agents for multimodal cellular imaging, J. Mater. Chem. 20 (44) (2010) 9895 9902. [36] B. Fadeel, A.E. Garcia-Bennett, Better safe than sorry: understanding the toxicological properties of inorganic nanoparticles manufactured for biomedical applications, Adv. Drug Deliv. Rev. 62 (3) (2010) 362 374. [37] A.C. Anselmo, S. Mitragotri, A review of clinical translation of inorganic nanoparticles, AAPS J. 17 (5) (2015) 1041 1054. [38] Q.-Y. Wang, Y.-J. Kang, Bioprobes based on aptamer and silica fluorescent nanoparticles for bacteria Salmonella typhimurium detection, Nanoscale Res. Lett. 11 (1) (2016) 150. [39] L. Pascual, et al., Oligonucleotide-capped mesoporous silica nanoparticles as DNA-responsive dye delivery systems for genomic DNA detection, Chem. Commun. 51 (8) (2015) 1414 1416. [40] H. Jo, J. Her, C. Ban, Dual aptamer-functionalized silica nanoparticles for the highly sensitive detection of breast cancer, Biosens. Bioelectron. 71 (2015) 129 136. [41] W. Stöber, A. Fink, E. Bohn, Controlled growth of monodisperse silica spheres in the micron size range, J. Colloid Interface Sci. 26 (1) (1968) 62 69. [42] L. Wang, W. Zhao, W. Tan, Bioconjugated silica nanoparticles: development and applications, Nano Res. 1 (2) (2008) 99 115. [43] Q. Deng, et al., Guanidinium functionalized superparamagnetic silica spheres for selective enrichment of phosphopeptides and intact phosphoproteins from complex mixtures, J. Mater. Chem. B 2 (8) (2014) 1048 1058. [44] V. Mamaeva, C. Sahlgren, M. Lindén, Mesoporous silica nanoparticles in medicine—recent advances, Adv. Drug Deliv. Rev. 65 (5) (2013) 689 702. [45] L. Wang, et al., Watching silica nanoparticles glow in the biological world, Anal. Chem. 78 (3) (2006) 646 654. [46] J.M. Rosenholm, C. Sahlgren, M. Lindén, Multifunctional mesoporous silica nanoparticles for combined therapeutic, diagnostic and targeted action in cancer treatment, Curr. Drug Targets 12 (8) (2011) 1166 1186. [47] L. Tang, J. Cheng, Nonporous silica nanoparticles for nanomedicine application, Nano Today 8 (3) (2013) 290 312. [48] X. Huang, M.A. El-Sayed, Gold nanoparticles: optical properties and implementations in cancer diagnosis and photothermal therapy, J. Adv. Res. 1 (1) (2010) 13 28. [49] P.K. Jain, K.S. Lee, I.H. El-Sayed, M.A. El-Sayed, Calculated absorption and scattering properties of gold nanoparticles of different size, shape, and composition: applications in biological imaging and biomedicine, J. Phys. Chem. B 110 (2006) 7238 7248. [50] E.E. Connor, J. Mwamuka, A. Gole, C.J. Murphy, M.D. Wyatt, Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity, Small 1 (3) (2005) 325 327. [51] P. Pedrosa, R. Vinhas, A. Fernandes, P.V. Baptista, Gold nanotheranostics: proof-of-concept or clinical tool?, Nanomaterials 5 (2015) 1853 1879. [52] M.A. El-Sayed, Some interesting properties of metals confined in time and nanometer space of different shapes, Acc. Chem. Res. 34 (4) (2001) 257 264. [53] R.S. Riley, E.S. Day, Gold nanoparticle-mediated photothermal therapy: applications and opportunities for multimodal cancer treatment, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 9 (4) (2017) e1449. [54] Nanospectra Biosciences

Information for Clinicians.

Chapter 4 • Clinical Translation of Nanomaterials

105

[55] S. Hassan, G. Prakash, A. Bal, S. Saghazadeh, Y. Shrike, A. Khademhosseini, Nano Today Evolution and clinical translation of drug delivery nanomaterials, Nano Today 15 (2017) 91 106. [56] H. Bin Na, I.C. Song, T. Hyeon, Inorganic nanoparticles for MRI contrast agents, Adv. Mater. 21 (21) (2009) 2133 2148. [57] A. Jordan, P. Wust, H. Fähling, W. John, A. Hinz, R. Felix, Inductive heating of ferrimagnetic particles and magnetic fluids: physical evaluation of their potential for hyperthermia, Int. J. Hyperthermia 9 (1) (1993) 51 68. [58] I. Hilger, R. Hiergeist, R. Hergt, K. Winnefeld, H. Schubert, W.A. Kaiser, Thermal ablation of tumors using magnetic nanoparticles: an in vivo feasibility study, Invest. Radiol. 37 (10) (2002) 580 586. [59] J.L. Gaglia, et al., Noninvasive mapping of pancreatic inflammation in recent-onset type-1 diabetes patients, Proc. Natl. Acad. Sci. U.S.A. 112 (7) (2015) 2139 2144. [60] M.S. Thu, et al., Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by MRI HHS public access, Nat. Med. 18 (3) (2012) 463 467. [61] C. Kaittanis, et al., Environment-responsive nanophores for therapy and treatment monitoring via molecular MRI quenching, Nat. Commun. 5 (2014) 3384. [62] P.B. Santhosh, N.P. Ulrih, Multifunctional superparamagnetic iron oxide nanoparticles: promising tools in cancer theranostics, Cancer Lett. 336 (1) (2013) 8 17. [63] R. Imani, et al., Multifunctional gadolinium-doped mesoporous TiO2 nanobeads: photoluminescence, enhanced spin relaxation, and reactive oxygen species photogeneration, beneficial for cancer diagnosis and treatment, Small 13 (20) (2017) 1700349. [64] S. Park, Acetone gas detection using TiO2 nanoparticles functionalized In2O3 nanowires for diagnosis of diabetes, J. Alloys Compd. 696 (696) (2017) 655 662. [65] A.M. Iga, J.H.P. Robertson, M.C. Winslet, A.M. Seifalian, Clinical potential of quantum dots, J. Biomed. Biotechnol. 2007 (10) (2007) 1 10. [66] S.J. Rosenthal, J.C. Chang, O. Kovtun, J.R. McBride, I.D. Tomlinson, Biocompatible quantum dots for biological applications, Chem. Biol. 18 (1) (2011) 10 24. [67] M. Bruchez, M. Moronne, P. Gin, S. Weiss, A.P. Alivisatos, Semiconductor nanocrystals as fluorescent biological labels, Science 281 (5385) (1998) 2013 2016. [68] A.M. Smith, H. Duan, A.M. Mohs, S. Nie, Bioconjugated quantum dots for in vivo molecular and cellular imaging, Adv. Drug Deliv. Rev. 60 (11) (2008) 1226 1240. [69] M.L. Etheridge, S.A. Campbell, A.G. Erdman, C.L. Haynes, S.M. Wolf, J. McCullough, The big picture on nanomedicine: the state of investigational and approved nanomedicine products, Nanomed. Nanotechnol. Biol. Med. 9 (1) (2013) 1 14. [70] L.O. Cinteza, Quantum dots in biomedical applications: advances and challenges, J. Nanophotonics 4 (1) (2010) 42503. [71] I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Mattoussi, Quantum dot bioconjugates for imaging, labelling and sensing, Nat. Mater. 4 (6) (2005) 435 446. [72] W. Jiang, E. Papa, H. Fischer, S. Mardyani, W.C.W. Chan, Semiconductor quantum dots as contrast agents for whole animal imaging, Trends Biotechnol. 22 (12) (2004) 607 609. [73] Z. Yun, D. Zhengtao, Y. Jiachang, T. Fangqiong, W. Qun, Using cadmium telluride quantum dots as a proton flux sensor and applying to detect H9 avian influenza virus, Anal. Biochem. 364 (2) (2007) 122 127. [74] S. Moffatt, Nanodiagnostics: a revolution in biomedical nanotechnology, MOJ Proteomics Bioinform. 3 (2) (2016). [75] J.A. Barreto, W. O’Malley, M. Kubeil, B. Graham, H. Stephan, L. Spiccia, Nanomaterials: applications in cancer imaging and therapy, Adv. Mater. 23 (12) (2011) H18 H40.

106

THERANOSTIC BIONANOMATERIALS

[76] NYC Population Facts, Department of City Planning. Available: ,https://www1.nyc.gov/site/planning/ data-maps/nyc-population/population-facts.page., 2018 (accessed 22.02.18). [77] S. Bhattacharyya, R.A. Kudgus, R. Bhattacharya, P. Mukherjee, Inorganic nanoparticles in cancer therapy, Pharm. Res. 28 (2) (2011) 237 259. [78] T. Kim, T. Hyeon, Applications of inorganic nanoparticles as therapeutic agents, Nanotechnology 25 (1) (2014) 12001. [79] D. Bobo, K.J. Robinson, J. Islam, K.J. Thurecht, S.R. Corrie, Nanoparticle-based medicines: a review of FDA-approved materials and clinical trials to date, Pharm. Res. 33 (10) (2016) 2373 2387. [80] C.L. Ventola, Progress in nanomedicine: approved and investigational nanodrugs, P T 42 (12) (2017) 742 755. [81] R. Gref, et al., Biodegradable long-circulating polymeric nanospheres, Science 263 (5153) (1994) 1600 1603. [82] C.-M.J. Hu, R.H. Fang, B.T. Luk, L. Zhang, Polymeric nanotherapeutics: clinical development and advances in stealth functionalization strategies, Nanoscale 6 (2014) 65 75. [83] S. Stolnik, L. Illum, S.S. Davis, Long circulating microparticulate drug carriers, Adv. Drug Deliv. Rev. 16 (2 3) (1995) 195 214. [84] J.M. Bliley, K.G. Marra, Polymeric biomaterials as tissue scaffolds, Stem Cell Biology and Tissue Engineering in Dental Sciences, Elsevier, 2015, pp. 149 161. [85] M. Ing, et al., Immunogenicity of long-lasting recombinant factor VIII products, Cell. Immunol. 301 (2016) 40 48. [86] J.L. Perry, et al., PEGylated PRINT nanoparticles: the impact of PEG density on protein binding, macrophage association, biodistribution, and pharmacokinetics, Nano Lett. 12 (10) (2012) 5304 5310. [87] Gref, et al., ‘Stealth’ corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption., Colloids Surf. B. Biointerfaces 18 (3 4) (2000) 301 313. [88] H. Xu, K.Q. Wang, Y.H. Deng, D.W. Chen, Effects of cleavable PEG-cholesterol derivatives on the accelerated blood clearance of PEGylated liposomes, Biomaterials 31 (17) (2010) 4757 4763. [89] J.K. Armstrong, et al., Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients, Cancer 110 (1) (2007) 103 111. [90] J. Kopeˇcek, P. Kopeˇcková, T. Minko, Z.-R. Lu, HPMA copolymer anticancer drug conjugates: design, activity, and mechanism of action, Eur. J. Pharm. Biopharm. 50 (1) (2000) 61 81. [91] D. Oupický, C. Konák, P.R. Dash, L.W. Seymour, K. Ulbrich, Effect of albumin and polyanion on the structure of DNA complexes with polycation containing hydrophilic nonionic block, Bioconjug. Chem. 10 (5) (1999) 764 772. [92] R. Duncan, M.J. Vicent, Do HPMA copolymer conjugates have a future as clinically useful nanomedicines? A critical overview of current status and future opportunities, Adv. Drug Deliv. Rev. 62 (2) (2010) 272 282. [93] L. Li, Q. Yang, Z. Zhou, J. Zhong, Y. Huang, Doxorubicin-loaded, charge reversible, folate modified HPMA copolymer conjugates for active cancer cell targeting, Biomaterials 35 (19) (2014) 5171 5187. [94] J.M. Caster, A.N. Patel, T. Zhang, A. Wang, Investigational nanomedicines in 2016: a review of nanotherapeutics currently undergoing clinical trials, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 9 (1) (2017) e1416. [95] J. Hrkach, et al., Preclinical development and clinical translation of a PSMA-targeted docetaxel nanoparticle with a differentiated pharmacological profile., Sci. Transl. Med. 4 (128) (2012) 128ra39. [96] J. Akimoto, M. Nakayama, T. Okano, Temperature-responsive polymeric micelles for optimizing drug targeting to solid tumors, J. Control. Release 193 (2014) 2 8.

Chapter 4 • Clinical Translation of Nanomaterials

107

[97] S. Keller, J.T. Wilson, G.I. Patilea, H.B. Kern, A.J. Convertine, P.S. Stayton, Neutral polymer micelle carriers with pH-responsive, endosome-releasing activity modulate antigen trafficking to enhance CD8 1 T cell responses, J. Control. Release 191 (2014) 24 33. [98] Y. Koyamatsu, T. Hirano, Y. Kakizawa, F. Okano, T. Takarada, M. Maeda, pH-responsive release of proteins from biocompatible and biodegradable reverse polymer micelles, J. Control. Release 173 (2014) 89 95. [99] M. Arseneault, C. Wafer, J.-F. Morin, Recent advances in click chemistry applied to dendrimer synthesis, Molecules 20 (12) (2015) 9263 9294. [100] E. Abbasi, et al., Dendrimers: synthesis, applications, and properties, Nanoscale Res. Lett. 9 (1) (2014) 247. [101] X. Cai, J. Hu, J. Xiao, Y. Cheng, Dendrimer and cancer: a patent review (2006 present), Expert Opin. Ther. Patents 23 (4) (2013) 515 529. [102] I. Singh, A.K. Rehni, R. Kalra, G. Joshi, M. Kumar, Dendrimers and their pharmaceutical applications a review, Pharmazie 63 (7) (2008) 491 496. [103] X.-X. Deng, F.-S. Du, Z.-C. Li, Combination of orthogonal ABB and ABC multicomponent reactions toward efficient divergent synthesis of dendrimers with structural diversity, ACS Macro Lett 3 (7) (2014) 667 670. [104] O. Kreye, D. Kugele, L. Faust, M.A.R. Meier, Divergent dendrimer synthesis via the passerini threecomponent reaction and olefin cross-metathesis, Macromol. Rapid Commun. 35 (3) (2014) 317 322. [105] D.J. Bharali, M. Khalil, M. Gurbuz, T.M. Simone, S.A. Mousa, Nanoparticles and cancer therapy: a concise review with emphasis on dendrimers, Int. J. Nanomed. 4 (2009) 1 7. [106] D.P. Otto, M.M. De Villiers, Poly(amidoamine) dendrimers as a pharmaceutical excipient. Are we there yet? J. Pharm. Sci. 107 (2018) 75 83. [107] W. Lohcharoenkal, L. Wang, Y.C. Chen, Y. Rojanasakul, Protein nanoparticles as drug delivery carriers for cancer therapy., Biomed Res. Int. 2014 (2014) 180549. [108] A.O. Elzoghby, W.M. Samy, N.A. Elgindy, Protein-based nanocarriers as promising drug and gene delivery systems, J. Control. Release 161 (1) (2012) 38 49. [109] M.R. Green, et al., Abraxanes, a novel Cremophors-free, albumin-bound particle form of paclitaxel for the treatment of advanced non-small-cell lung cancer, Ann. Oncol 17 (8) (2006) 1263 1268. [110] M.J. Vicent, R. Duncan, Polymer conjugates: nanosized medicines for treating cancer, Trends Biotechnol. 24 (1) (2006) 39 47. [111] M. Santoro, A.M. Tatara, A.G. Mikos, Gelatin carriers for drug and cell delivery in tissue engineering, J. Control. Release 190 (2014) 210 218. [112] J. Madan, R.S. Pandey, U.K. Jain, O.P. Katare, R. Aneja, A. Katyal, Sterically stabilized gelatin microassemblies of noscapine enhance cytotoxicity, apoptosis and drug delivery in lung cancer cells, Colloids Surf. B Biointerfaces 107 (2013) 235 244. [113] S. Kommareddy, M. Amiji, Poly(ethylene glycol) modified thiolated gelatin nanoparticles for glutathione-responsive intracellular DNA delivery, Nanomed. Nanotechnol. Biol. Med. 3 (1) (2007) 32 42. [114] J.C. Kraft, J.P. Freeling, Z. Wang, R.J.Y. Ho, Emerging research and clinical development trends of liposome and lipid nanoparticle drug delivery systems, J. Pharm. Sci. 103 (1) (2014) 29 52. [115] A. Meyerhoff, U.S. Food and Drug Administration approval of AmBisome (liposomal amphotericin B) for Treatment of visceral leishmaniasis, Clin. Infect. Dis. 28 (1) (1999) 42 48. [116] K. Hetter, Can this cream remove your tattoo? CNN, CNN. Available: ,http://edition.cnn.com/2015/ 02/19/living/feat-new-tattoo-removal-cream/index.html., 2015 (accessed 08.02.18).

108

THERANOSTIC BIONANOMATERIALS

[117] J. Gallagher, Heat-triggered ‘grenades’ hit cancer - BBC News, BBC News. Available: ,http://www.bbc. com/news/health-34667804., 2015 (accessed: 08.02.18). [118] S. Batzri, E.D. Korn, Single bilayer liposomes prepared without sonication., Biochim. Biophys. Acta 298 (4) (1973) 1015 1019. [119] F. Szoka, D. Papahadjopoulos, D. Papahadjopoulos, Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation., Proc. Natl. Acad. Sci. U.S.A. 75 (9) (1978) 4194 4198. [120] F. Frézard, M.S. Michalick, C.F. Soares, C. Demicheli, Novel methods for the encapsulation of meglumine antimoniate into liposomes., Braz. J. Med. Biol. Res. Rev. Bras. Pesqui. med. Biol. 33 (7) (2000) 841 846. [121] Y.P. Patil, S. Jadhav, Novel methods for liposome preparation, Chem. Phys. Lipids 177 (2014) 8 18. [122] I.E. Santo, R. Campardelli, E.C. Albuquerque, S.V. de Melo, G. Della Porta, E. Reverchon, Liposomes preparation using a supercritical fluid assisted continuous process, Chem. Eng. J. 249 (2014) 153 159. [123] K. Elerˇsiˇc, J.I. Pavliˇc, A. Igliˇc, A. Vesel, M. Mozetiˇc, Electric-field controlled liposome formation with embedded superparamagnetic iron oxide nanoparticles, Chem. Phys. Lipids 165 (1) (2012) 120 124. [124] E. Kastner, V. Verma, D. Lowry, Y. Perrie, Microfluidic-controlled manufacture of liposomes for the solubilisation of a poorly water soluble drug, Int. J. Pharm. 485 (1 2) (2015) 122 130. [125] F. Kong, et al., Inhibition of multidrug resistance of cancer cells by co-delivery of DNA nanostructures and drugs using porous silicon nanoparticles@giant liposomes, Adv. Funct. Mater. 25 (22) (2015) 3330 3340. [126] N. Li, et al., Development of targeting lonidamine liposomes that circumvent drug-resistant cancer by acting on mitochondrial signaling pathways, Biomaterials 34 (13) (2013) 3366 3380. [127] H.B. Ruttala, Y.T. Ko, Liposomal co-delivery of curcumin and albumin/paclitaxel nanoparticle for enhanced synergistic antitumor efficacy, Colloids Surf. B Biointerfaces 128 (2015) 419 426. [128] Z. Peng, C. Wang, E. Fang, X. Lu, G. Wang, Q. Tong, Co-delivery of doxorubicin and SATB1 shRNA by thermosensitive magnetic cationic liposomes for gastric cancer therapy, PLoS One 9 (3) (2014) e92924. [129] D. Liu, et al., Quercetin deformable liposome: preparation and efficacy against ultraviolet B induced skin damages in vitro and in vivo, J. Photochem. Photobiol. B Biol. 127 (2013) 8 17. [130] W.-Z. Li, et al., Propylene glycol-embodying deformable liposomes as a novel drug delivery carrier for vaginal fibrauretine delivery applications, J. Control. Release 226 (2016) 107 114. [131] R. Kulkarni, J.D. Yadav, K.A. Vaidya, P.P. Gandhi, Transferosomes: an emerging tool for transdermal drug delivery, Int. J. Pharm. Sci. Res. 2 (4) (2011) 735 741. [132] M. Castillo, 371 million people have diabetes globally, about half undiagnosed - CBS News, CBS News. Available: ,https://www.cbsnews.com/news/371-million-people-have-diabetes-globally-about-halfundiagnosed/., 2012 (accessed 08.02.18). [133] G. Chen, et al., Deformable liposomes by reverse-phase evaporation method for an enhanced skin delivery of (1)-catechin, Drug Dev. Ind. Pharm. 40 (2) (2014) 260 265. [134] R. Chen, et al., Ultradeformable liposomes: a novel vesicular carrier for enhanced transdermal delivery of procyanidins: effect of surfactants on the formation, stability, and transdermal delivery, AAPS PharmSciTech 18 (5) (2017) 1823 1832. [135] B. Kneidl, M. Peller, G. Winter, L.H. Lindner, M. Hossann, Thermosensitive liposomal drug delivery systems: state of the art review, Int. J. Nanomed. 9 (2014) 4387 4398. [136] M.S. Muthu, S.-S. Feng, Theranostic liposomes for cancer diagnosis and treatment: current development and pre-clinical success, Expert Opin. Drug Deliv. 10 (2) (2013) 151 155.

Chapter 4 • Clinical Translation of Nanomaterials

109

[137] S. Dünnhaupt, et al., S-protected thiolated chitosan for oral delivery of hydrophilic macromolecules: evaluation of permeation enhancing and efflux pump inhibitory properties, Mol. Pharm. 9 (5) (2012) 1331 1341. [138] K. Senchi, S. Matsunaga, H. Hasegawa, H. Kimura, A. Ryo, Development of oligomannose-coated liposome-based nasal vaccine against human parainfluenza virus type 3, Front. Microbiol. 4 (2013) 346. [139] G.T. Noble, J.F. Stefanick, J.D. Ashley, T. Kiziltepe, B. Bilgicer, Ligand-targeted liposome design: challenges and fundamental considerations, Trends Biotechnol. 32 (1) (2014) 32 45. [140] M.B. Yatvin, J.N. Weinstein, W.H. Dennis, R. Blumenthal, Design of liposomes for enhanced local release of drugs by hyperthermia, Science 202 (4374) (1978) 1290 1293. [141] S.M. Park, et al., Novel temperature-triggered liposome with high stability: formulation, in vitro evaluation, and in vivo study combined with high-intensity focused ultrasound (HIFU), J. Control. Release 170 (3) (2013) 373 379. [142] M. Vila-Caballer, et al., A pH-sensitive stearoyl-PEG-poly(methacryloyl sulfadimethoxine)-decorated liposome system for protein delivery: an application for bladder cancer treatment, J. Control. Release 238 (2016) 31 42. [143] L. Bana, et al., Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood brain-barrier: implications for therapy of Alzheimer disease, Nanomed. Nanotechnol. Biol. Med 10 (7) (2014) 1583 1590. [144] N. Kamaly, Z. Xiao, P.M. Valencia, A.F. Radovic-Moreno, O.C. Farokhzad, Targeted polymeric therapeutic nanoparticles: design, development and clinical translation, Chem. Soc. Rev. 41 (7) (2012) 2971. [145] L. Zhang, F. Gu, J. Chan, A. Wang, R. Langer, O. Farokhzad, Nanoparticles in medicine: therapeutic applications and developments, Clin. Pharmacol. Ther. 83 (5) (2008) 761 769. [146] X. Ma, Y. Zhao, X.-J. Liang, Theranostic nanoparticles engineered for clinic and pharmaceutics, Acc. Chem. Res. 44 (10) (2011) 1114 1122. [147] J.H. Ryu, et al., Theranostic nanoparticles for future personalized medicine, J. Control. Release 190 (2014) 477 484. [148] S.M. Janib, A.S. Moses, J.A. MacKay, Imaging and drug delivery using theranostic nanoparticles, Adv. Drug Deliv. Rev. 62 (11) (2010) 1052 1063. [149] J. Lannutti, D. Reneker, T. Ma, D. Tomasko, D. Farson, Electrospinning for tissue engineering scaffolds, Mater. Sci. Eng. C 27 (3) (2007) 504 509. [150] R.M. Nerem, A. Sambanis, Tissue engineering: from biology to biological substitutes, Tissue Eng. 1 (1) (1995) 3 13. [151] S.J. Hollister, Porous scaffold design for tissue engineering, Nat. Mater. 4 (7) (2005) 518 524. [152] S.C. Cox, J.A. Thornby, G.J. Gibbons, M.A. Williams, K.K. Mallick, 3D printing of porous hydroxyapatite scaffolds intended for use in bone tissue engineering applications, Mater. Sci. Eng. C 47 (2015) 237 247. [153] M.A. Fernandez-Yague, S.A. Abbah, L. McNamara, D.I. Zeugolis, A. Pandit, M.J. Biggs, Biomimetic approaches in bone tissue engineering: integrating biological and physicomechanical strategies, Adv. Drug Deliv. Rev. 84 (2015) 1 29. [154] M. Manassero, A. Decambron, N. Guillemin, H. Petite, R. Bizios, V. Viateau, Coral scaffolds in bone tissue engineering and bone regeneration, The Cnidaria, Past, Present and Future, Springer International Publishing, Cham, 2016, pp. 691 714. [155] C.M. Murphy, et al., A collagen hydroxyapatite scaffold allows for binding and co-delivery of recombinant bone morphogenetic proteins and bisphosphonates, Acta Biomater. 10 (5) (2014) 2250 2258.

110

THERANOSTIC BIONANOMATERIALS

[156] B. Guo, P.X. Ma, Synthetic biodegradable functional polymers for tissue engineering: a brief review, Sci. China Chem. 57 (4) (2014) 490 500. [157] S.R. Dutta, D. Passi, P. Singh, A. Bhuibhar, Ceramic and non-ceramic hydroxyapatite as a bone graft material: a brief review, Irish J. Med. Sci. (1971 -) 184 (1) (2015) 101 106. [158] H.-L. Kim, et al., Preparation and characterization of nano-sized hydroxyapatite/alginate/chitosan composite scaffolds for bone tissue engineering, Mater. Sci. Eng. C 54 (2015) 20 25. [159] K.F. Leong, C.M. Cheah, C.K. Chua, Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs, Biomaterials 24 (13) (2003) 2363 2378. [160] D.W. Hutmacher, Scaffold design and fabrication technologies for engineering tissues—state of the art and future perspectives, J. Biomater. Sci. Polym. Ed. 12 (1) (2001) 107 124. [161] M.E. Hoque, Y.L. Chuan, I. Pashby, Extrusion based rapid prototyping technique: an advanced platform for tissue engineering scaffold fabrication, Biopolymers 97 (2) (2012) 83 93. [162] C.T. Johnson, A.J. García, Scaffold-based anti-infection strategies in bone repair, Ann. Biomed. Eng. 43 (3) (2015) 515 528. [163] I. Grulova, et al., Delivery of alginate scaffold releasing two trophic factors for spinal cord injury repair., Sci. Rep. 5 (2015) 13702. [164] X. Shen, et al., Sequential and sustained release of SDF-1 and BMP-2 from silk fibroinnanohydroxyapatite scaffold for the enhancement of bone regeneration, Biomaterials 106 (2016) 205 216. [165] L. Wang, L. Cao, J. Shansky, Z. Wang, D. Mooney, H. Vandenburgh, Minimally invasive approach to the repair of injured skeletal muscle with a shape-memory scaffold, Mol. Ther. 22 (8) (2014) 1441 1449. [166] A. Khademhosseini, R. Langer, A decade of progress in tissue engineering, Nat. Protoc. 11 (10) (2016) 1775 1781. [167] O. Wichterle, D. Lím, Hydrophilic gels for biological use, Nature 185 (4706) (1960) 117 118. [168] E.M. Ahmed, Hydrogel: preparation, characterization, and applications: a review, J. Adv. Res. 6 (2) (2015) 105 121. [169] N.A. Peppas, P. Bures, W. Leobandung, H. Ichikawa, Hydrogels in pharmaceutical formulations, Eur. J. Pharm. Biopharm. 50 (1) (2000) 27 46. [170] A.S. Hoffman, Hydrogels for biomedical applications, Adv. Drug Deliv. Rev. 64 (2012) 18 23. [171] T.R. Hoare, D.S. Kohane, Hydrogels in drug delivery: progress and challenges, Polymer (Guildf). 49 (8) (2008) 1993 2007. [172] A. Harada, J. Li, M. Kamachi, Double-stranded inclusion complexes of cyclodextrin threaded on poly (ethylene glycol), Nature 370 (6485) (1994) 126 128. [173] J. Li, X. Li, X. Ni, X. Wang, H. Li, K.W. Leong, Self-assembled supramolecular hydrogels formed by biodegradable PEO PHB PEO triblock copolymers and α-cyclodextrin for controlled drug delivery, Biomaterials 27 (22) (2006) 4132 4140. [174] E. Caló, V.V. Khutoryanskiy, Biomedical applications of hydrogels: a review of patents and commercial products, Eur. Polym. J. 65 (2015) 252 267. [175] M. Ebara, et al., Smart Biomaterials, Springer, 2014. [176] M.F. Akhtar, M. Hanif, N.M. Ranjha, Methods of synthesis of hydrogels . . . a review, Saudi Pharm. J. 24 (5) (2016) 554 559. [177] L. Li, B. Yan, J. Yang, L. Chen, H. Zeng, Novel mussel-inspired injectable self-healing hydrogel with anti-biofouling property, Adv. Mater. 27 (7) (2015) 1294 1299. [178] X. Zhao, et al., Injectable stem cell-laden photocrosslinkable microspheres fabricated using microfluidics for rapid generation of osteogenic tissue constructs, Adv. Funct. Mater. 26 (17) (2016) 2809 2819.

Chapter 4 • Clinical Translation of Nanomaterials

111

[179] F. Bierbrauer, Hydrogel Drug Delivery: Diffusion Models. ,www.bierbrauerf.weebly.com. [180] D.L. Taylor, M. In Het Panhuis, Self-healing hydrogels, Adv. Mater. 28 (41) (2016) 9060 9093. [181] X. Sun, et al., Electrospun photocrosslinkable hydrogel fibrous scaffolds for rapid in vivo vascularized skin flap regeneration, Adv. Funct. Mater. 27 (2) (2017) 1604617. [182] S. Hassan, et al., Evolution and clinical translation of drug delivery nanomaterials, Nano Today 15 (2017) 91 106. [183] T. Lammers, Smart drug delivery systems: back to the future vs. clinical reality, Int. J. Pharm. 454 (1) (2013) 527 529. [184] J.I. Hare, T. Lammers, M.B. Ashford, S. Puri, G. Storm, S.T. Barry, Challenges and strategies in anticancer nanomedicine development: an industry perspective, Adv. Drug Deliv. Rev. 108 (2017) 25 38. [185] X. Li, et al., Effects of physicochemical properties of nanomaterials on their toxicity, J. Biomed. Mater. Res., A 103 (7) (2015) 2499 2507. [186] Y. Zhang, et al., Toxicity and efficacy of carbon nanotubes and graphene: the utility of carbon-based nanoparticles in nanomedicine, Drug Metab. Rev. 46 (2) (2014) 232 246. [187] I.-Y. Kim, E. Joachim, H. Choi, K. Kim, Toxicity of silica nanoparticles depends on size, dose, and cell type, Nanomed. Nanotechnol. Biol. Med. 11 (6) (2015) 1407 1416. [188] I. Fratoddi, I. Venditti, C. Cametti, M.V. Russo, How toxic are gold nanoparticles? The state-of-the-art, Nano Res. 8 (6) (2015) 1771 1799. [189] H. Arami, A. Khandhar, D. Liggitt, K.M. Krishnan, In vivo delivery, pharmacokinetics, biodistribution and toxicity of iron oxide nanoparticles, Chem. Soc. Rev. 44 (23) (2015) 8576 8607. [190] Y. Min, J.M. Caster, M.J. Eblan, A.Z. Wang, Clinical translation of nanomedicine, Chem. Rev. 115 (19) (2015) 11147 11190.

5 Synthetic Receptors With Bioaffinity for Biomedical Applications Wei Chen1,2, Yue Ma3, Guoqing Pan1 1

INSTITUTE FOR ADVANCED MA TERIALS, SCHO OL OF MA TERIAL S S CIENCE AN D ENGINEERING, JIANGSU UNIVERSITY, ZHENJ IANG, P.R. CHINA 2 C OLLE GE OF CHE MIC AL AND E NV I R O N ME N T A L E N G I NE E R I N G , S HA N D O N G U N IVERSITY OF SCIENCE AND TECHNOLOGY, QINGDAO, P .R. C HINA 3 SCHOOL OF CHEMISTRY AND CHEMICAL ENGINE ERING, JIANGSU UNIVERSITY, ZHENJIANG, P.R. CHINA

5.1 Introduction The specific interactions (i.e., molecular recognitions) between receptors at the cell membrane and ligands at the extracellular matrix (ECM) are crucial in various cellular processes [1]. The occurrence of these molecular recognitions as a consequence of ECM remodeling gives rise to specific cell-signaling and intracellular cascades. Therefore the natural receptor
ligand interactions are central in physiology and pathological processes. From the point of view of materials science, the specific binding of natural receptors to targeted ligands also shows great promise in the design of biomaterials with advanced affinity [2]. Despite the successful development of a plethora of biomaterials or biomedical devices based on natural receptor
ligand interactions in the last decade, natural species like proteins or DNAs still have inherent drawbacks [3,4]. First, the chemical and physical stability and shelf life of natural receptors or antibodies are limited, which restricts their applications in nonphysiological environments. Second, it is expensive, time-consuming, and laborintensive to isolate and purify them from nature or by biochemical synthesis. Finally, practical requirements in materials science are more extensive than the limited yield, functions, and diversity in currently available molecular recognition in nature. As a result, an advanced material design with more durable and robust receptor-like substitutions is sought [5]. It was found that a chemical combination of multiple noncovalent or reversible covalent interactions with spatial and functional complementarity helps create synthetic receptors with molecular recognition properties similar to those of natural ones [4,6]. With the increasing demand for multiple functions and properties in the development of advanced biomaterials, chemically designed substitutes with receptor-like properties (i.e., synthetic receptors) are highly sought after [7
10]. After decades of development, several classic strategies, such as molecular imprinting, affinity screening, dynamic combinatorial chemistry, and de novo structure-based design strategies, have been exploited for the design and preparation of Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00005-5 © 2019 Elsevier Inc. All rights reserved.

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receptor-like substitutes [2]. As natural receptor mimics, these synthetic receptor-like substitutes are more durably stable compared to natural ones, thus showing great potential in affinity-related materials science, particularly for biomedical-related applications due to their highly biomimetic nature. Preparations of synthetic receptors based on molecular imprinting and affinity screening have drawn great attention in biomedical applications because of their simplicity, low cost, and mild conditions (Fig. 5
1) [2]. The molecular imprinting process involves the self-assembly of template molecules and functional monomers via noncovalent or reversible covalent bonds; the resulting complexes are subsequently copolymerized with a suitable cross-linker. After removing the templates from the cross-linked polymer network, molecular recognition sites, complementary in shape, size, and functionality to the template molecules, are formed in the molecularly imprinted polymers (MIPs) [11,12]. In principle, molecular imprinting allows rapid and inexpensive generation of synthetic receptors from nearly all target molecules, for example, small molecules, peptides, proteins, and DNA. In contrast, an affinity screening strategy is simpler; the mechanism is based on optimizing the selection and proportion of various functional monomers that are predisposed toward favorable interactions with the target molecules. After screening from a library of synthetic polymer nanoparticles (NPs) or linear copolymer incorporating a diverse pool of functional monomers, receptor-like candidates with high affinity and selectivity to targeted biomacromolecules can be readily obtained [13]. Thus the intrinsic features of molecular imprinting and affinity screening strategy fit well with the preparation of synthetic receptors for bio-related molecular recognition and subsequently biomedical applications.

FIGURE 5–1 Summary of the two main approaches to preparation of synthetic receptors: (A) molecular imprinting process and (B) affinity screening strategy. Reproduced with permission from 2014 Springer Nature.

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In this chapter, the latest biomedical advances of synthetic receptors with biomolecular affinity based on the molecular imprinting and affinity screening strategy will be emphatically highlighted. Other than the preparation of synthetic receptors with biomolecular affinity, we will also review and discuss the representative and emerging applications of these synthetic receptors, such as detoxification, bacteriostasis, bio-imaging, cancer therapy, and cell isolation. At the end of the chapter, we give a perspective on this field, in particular, the future development of synthetic receptors for potential bio-applications. We hope this chapter will provide researchers with a panoramic view of synthetic receptors for biomedicines, and inspire them to develop new methods and materials for advanced applications in various biological and medical fields.

5.2 Synthetic Receptors for Biomedicines 5.2.1 Toxin Neutralization Toxins are peptides or proteins secreted by bacteria or animals which are virulence factors that cause an immediate threat to human life. An appropriate antidote is necessary to mitigate this threat. Like antivenom, with active components of polyclonal antibodies to neutralize venom, most antidotes are biological products [14]. Polyclonal antibodies are purified from animal blood after eliciting immune responses by injecting venom [15]. As antivenoms are costly and time-consuming to generate, inexpensive antivenom therapeutic approaches are highly sought after. Synthetic receptors with antibody-like properties show great potential for this purpose [16]. The development of material interfacial bio-absorption revealed that synthetic polymer receptors exhibit potential applications as alternatives to biological antidotes [17,18]. Commonly, synthetic polymers with uniform size and chemical components are synthesized through precipitation or emulsion polymerizations in water solution, and are purified by dialyzing the resulting colloidal suspension against excess water. The chemical compositions of these copolymer systems are comprised of carbon backbones with randomly distributed functional side chains by radical polymerization of different functional monomers. To obtain random stoichiometric incorporation corresponding to the feed ratio, the functional monomers should be either acrylamide or methylacrylamide due to their similar reactivity ratios. After screening from a library of synthetic polymers incorporating a diverse pool of functional monomers, receptor-like candidates with high affinity and selectivity to targeted peptide or proteins can be readily obtained. Hoshino et al. reported the synthesis of multifunctional polymer NPs, with antibody-like affinity, to melittin, a biological toxin, via the affinity screening method (Fig. 5
2) [17]. A small library of synthetic polymer NPs was prepared by copolymerizing functional monomers, including N-isopropylacrylamide (NIPAm), N-tert-butylacrylamide (TBAm), acrylic acid (AAc), N-3-aminopropyl methacrylamide, and N,N0 -methylenebis(acrylamide) (BIS) as a dominate monomer, hydrophobic monomer, negatively charged monomer, positively charged functional monomers, and cross-linker, respectively. The monomer ratio and

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FIGURE 5–2 (A) Neutralization constants of NPs obtained from hemolytic toxicity neutralization assay. (B) Apparent binding constant between melittin and NPs. (C) Amount of NP aggregation that formed by incubation with mouse plasma followed by centrifugation. (D) In vivo imaging of fluorescent-labeled melittin after intravenous injection. NP4 or NP9 were injected 20 s after the injection of melittin. (E) Fluorescent ex vivo images of fluorescent-labeled melittin of mice followed with and without NP9. (F) Biodistribution of 14C-labeled NP9 in mice 30 min after administration. (G) Fluorescence histology images of a liver 70 min after injection of fluorescent-labeled melittin and NP9. Green; fluoroscein-NP9, red; fluorescent-labeled melittin, yellow; merged. Scale bars: 25 μm. NP, Nanoparticle. Reproduced with permission from 2012 PNAS.

particle size were optimized and screened to have high affinity and selectivity to melittin. The affinity of the NPs was evaluated by quartz crystal microbalance (QCM); those with 40% AAc and 40% TBAm are the most attracted to melittin peptide. Furthermore, optimized receptor-like NPs could be directly used to neutralize melittin in a mice model in vivo; their in vivo toxin neutralization capacity is related to the peptide-binding affinity and capacity. In vivo fluorescent imaging experiments further revealed that the receptor-like NPs were accumulated in the liver after removing toxic peptide from blood (Fig. 5
2), indicating the NP receptors were recognized by macrophages in the liver similarly to other nano-objects. The study suggested that the synthetic NP receptors with high affinity toward melittin could efficiently remove toxin peptide from the blood followed by the macrophage uptake and liver detoxification [18]. In another study, Weisman et al. designed another synthetic NP receptor to bind and neutralize toxic peptide phenol-soluble modulin α3 (PSMα3) by the affinity screening method [19]. Besides TBAm and AAc, a hydrophobic aromatic monomer pentafluorophenyl acrylamide was chosen as the functional monomers because PSMα3 peptide is amphiphilic

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and positively charged at physiological pH. The hemolytic assay and QCM result showed that incorporating monomers in NP receptors leads to improved affinity to PSMα3. Obviously, the optimized NP receptors showed excellent property to neutralize the toxin peptide in vitro; the neutralization was not affected by serum proteins with similar nonspecific affinity, mainly due to the high specificity to PSMα3. Inspired by the success of in vivo toxin neutralization using the synthetic NP receptors mentioned earlier, O’Brien et al. optimized another synthetic NP receptor capable of sequestering and neutralizing venomous phospholipase A2 (PLA2). In this paper, AAc, N-phenylacrylamide, and NIPAm were chosen as functional monomers. The synthetic NPs with optimized chemical compositions exhibited high selectivity to venomous PLA2 over abundant serum proteins [20]. Other than the above cross-linked polymeric receptors (i.e., the NPs), Wada et al. reported the neutralization of the toxic peptide using multifunctional linear polymers (LPs). The linear receptor-like polymers, containing tert-butyl group and carboxylic acids, were prepared by reversible addition
fragmentation chain transfer polymerization (Fig. 5
3). The binding capacity of LPs to melittin was higher than NPs with the same combination of functional groups, indicating the importance of the polymer chain flexibility to binding capacity and affinity [21]. Furthermore, they investigated the minimization of molecular weight and density of functional units of the LPs to achieve specific binding affinity to the toxic peptide, which will be a valuable method to get “plastic aptamers” with strong binding affinity to target peptides [22].

FIGURE 5–3 (A) Crystal structure and amino acid sequence of melittin. (B) Schematic of the binding between linear polymer and melittin. (C) Preparation of multifunctional LPs. LP, Linear polymer. Reproduced with permission from 2015 Royal Society of Chemistry.

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In fact, the molecular imprinting method was used earlier than the affinity screening method for the design of synthetic receptors to neutralize and clear up toxic peptides. In 2008, Hoshino et al. synthesized a melittin-imprinted NP using NIPAm, TBAm, and AAc as the functional monomers, BIS as cross-linker, and the melittin peptide as template molecules, respectively (Fig. 5
4) [23,24]. The molecular imprinting process can create melittin binding sites on the synthetic receptors without tedious screening procedures. The authors finally obtained protein-sized MIPNPs with a binding affinity and selectivity toward melittin comparable to those of natural antibodies. Fluorescent imaging revealed that the distribution of melittin in vivo was obviously reduced by post administration of the MIPNPs; ex vivo results demonstrated that the melittin peptide was efficiently cleared from blood and accumulated in the liver. It can be concluded that the MIP-based synthetic receptors are likewise capable of capturing cytotoxic peptide melittin in the bloodstream, showing the medical potential for detoxification. Taken together, the above studies demonstrated that the affinity screening and molecular imprinting method could provide guidelines for the synthesis of synthetic receptors against a number of toxic proteins and peptides. Different from a molecular imprinting strategy that requires a target template for receptor synthesis, the affinity screening strategy mainly relies on adjusting the chemical composition. Thus it is very promising to massively exploit synthetic receptors for bio-recognition. Also, by employing diverse functional monomers and optimizing the combinations and ratios of monomers, synthetic receptors can be used as robust antidotes with the capabilities of capturing and neutralizing a wide range of target

FIGURE 5–4 (A) Monomers used for NP synthesis. (B) Solution-phase AFM images of MIPNPs. (C) Amino acid sequence of melittin. (D) Schematic of the preparation of MIPNPs. (E) Fluorescent images of Cyt5-labeled melittin 15 min after injection; MIPNPs were injected 20 s after the injection of melittin (right). NP, Nanoparticle. Reproduced with permission from 2010 American Chemical Society.

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toxins in vivo. These advantages indicate the promise of an affinity screening strategy in clinical broad-spectrum toxin sequestration and neutralization.

5.2.2 Bacteria Inhibition In addition to detoxification, a similar concept based on molecularly imprinted receptors has also been used to inhibit bacterial multiresistance by extracting an antibiotic enzyme, β-lactamase, from bacterial secretions (Fig. 5
5) [25]. As is known, the expression of β-lactamase is the most pervasive resistance mechanism employed by bacteria, because the enzyme can hydrolyze the β-lactam ring to deactivate antibiotics, thus providing multiresistant bacteria to β-lactam antibiotics [26]. In their study, a thermo-responsive imprinted hydrogel was prepared with excellent molecular recognition toward β-lactamase. Therefore the β-lactamase-imprinted hydrogel could initially trap β-lactamase excreted by drugresistant bacteria, thus making the bacteria sensitive to antibiotics and improving antibacterial activity. In this case, it could act as an adjuvant to enhance the efficacy of antibiotics against drug-resistant bacteria. Indeed, the resultant MIPs showed excellent adsorption

FIGURE 5–5 (A) Fabrication of thermo-responsive imprinted hydrogel using β-lactamase as template. (B) Scheme illustration of the thermo-responsive imprinted hydrogel for the treatment of antibiotic-resistant bacteria. (C) Binding isotherms of the IP and NP hydrogel toward β-lactamase at 37 C. (D) Binding isotherms of the IP and NP hydrogel at 20 C toward β-lactamase. (E) Selective adsorption of β-lactamase by the IP and NP hydrogel. (F) Viability analysis of bacteria samples in the presence of IP hydrogel. (G) Wounds of mice after 1 and 3 days of therapy. The mice samples 1
5 were treated with phosphate-buffered saline (PBS) buffer, the IP hydrogel, penicillin G, the NP hydrogel 1 penicillin G, and the IP hydrogel 1 penicillin G, respectively. (H) The bacteria separated from wound tissue were cultured on agar plates. Insets are the wound tissue. (I) Number of bacteria in the wound tissue of each sample. NP, Nanoparticle. Reproduced with permission from 2016 Wiley-VCH.

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capacity and selectivity to β-lactamase at 37 C. In contrast, the affinity toward β-lactamase dramatically decreased at 20 C. This result indicated that the “imprinted sites” on the hydrogel could be reversibly abolished with a temperature stimulus, which resulted in the reactivation of β-lactamase to degrade antibiotic residues. A methicillin-resistant Staphylococcus aureus (MRSA) bacteria was used to evaluate the ability of MIP hydrogel for antibacterial applications. In vitro experiments showed that the β-lactam antibiotic penicillin G exhibited low inhibition of MRSA, while the bacterial viability was decreased by approximately 80% in the presence of the imprinted-polymer (IP) hydrogel. In addition, the authors found that the MIP hydrogel is the most effective in wound antibacterial therapy. This work indicated that MIP-based synthetic receptors exhibit great promise in multifunctional biomedical applications, because they can be applied for detoxification and efficient removal of specific harmful secretions in the biosystem. Recently, Motib et al. developed a new type of linear MIP (LMIP) with affinity toward PhrA, a signaling peptide, as an antiinfective agent (Fig. 5
6) [27]. The LMIP was prepared with AAc, N-(3-aminopropyl)methacrylamide, acrylamide, and TBAm as functional monomers to establish hydrogen bonds, ionic interactions with the target peptide. Unlike traditional molecular imprinting approaches, no cross-linker was used in this study. In addition, computer modeling was used to predict the possible monomer sequence of the LMIP. As is known, bacteria can communicate with each other to modulate behaviors through the production and detection of signaling molecules, a mechanism called quorum sensing (QS) [28]. The TprA receptor and its signaling peptide PhrA are important for pneumococcal growth on galactose and mannose, as well as on mucin [29]. Due to the specific binding ability of LMIP to PhrA assessed by surface plasmon resonance (SPR), the LMIP was incubated with Pneumococcus to determine whether it could block the TprA receptor, attenuate the induction of β-galactosidase activity and decrease pneumococcal growth on galactose. As expected, in the presence of LMIP, the β-galactosidase activity was significantly lower (by 1.8-fold) compared to induction by PhrA10 alone, demonstrating the efficient inhibition of promoter activation by PhrA10. The impact of LMIP-PhrA on virulence was further confirmed by in vivo evaluation against lethal microbial challenge in a mouse model of pneumococcal pneumonia. Despite receiving a higher dose after 24 h, the cohort that received the inoculum together with LMIP-PhrA had lower blood counts than the cohort that received the dose in PBS, indicating that LMIP-PhrA prevented the translocation of pneumococci from lungs to blood. These results demonstrated that the LMIP could interfere with QS signals in high selectivity, and curtailing the phenotypic manifestation of this system both in vitro and in vivo. In the future, the LMIPs would become potentially antiinfective against pathogenic bacteria. In addition, Ma et al. demonstrated the feasibility of using the same mechanism to interrupt bacterial QS and further inhibit biofilm formation [30]. The amount and type of functional monomers, itaconic acid (IA) and 2-hydroxyethyl methacrylate (HEMA), being used to synthesize MIPs were optimized to achieve higher adsorption capacity and affinity. In this work, a prototypical QS autoinducer N-(3-oxododecanoyl)-L-homoserine lactone(3-oxo-C12-AHL) [31] was chosen as the template. Compared with IA-based MIPs, the HEMA-based MIPs exhibited

Chapter 5 • Synthetic Receptors With Bioaffinity for Biomedical Applications

FIGURE 5–6 (A) Computational modeling of the molecular complex between the LMIP fragments and the C-terminal end of the PhrA peptide. (B) The affinity of LMIP-PhrA to the PhrA10 peptide at different concentrations (SPR sensorgrams). (C) The effect of LMIP-PhrA/PhrA10 binding on to TprA receptor. (D) Phenotypic evaluation of the LMIP. (E) Phenotypic evaluation of the growth assay. LMIP, Linear molecularly imprinted polymer. Reproduced with permission from 2017 Wiley-VCH.

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higher adsorption capacity. Moreover, the formation of Pseudomonas aeruginosa biofilm on HEMA-based MIPs was reduced by B65% because of a reduced bacterial attachment on cell repellent poly(HEMA). Further study found that the HEMA-based MIPs showed antimicrobial activity on planktonic cells. Therefore MIPs-based synthetic receptors can be potentially used as alternative antiinfective and biofilm intervention agents in clinical settings and food processing.

5.2.3 Biomedical Imaging Cell imaging is of great interest in the visualization of specific cells or tissues and has tremendous potential in early disease diagnosis, such as for the cancers [32]. Moreover, imaging technologies and new targeted therapies could evaluate the therapeutic effectiveness long before morphological changes. Therefore intelligent materials that could recognize tumor cell or tumor-specific markers are urgently needed [33]. Synthetic receptors with specific affinity to biomolecules, like cell membrane glycans, proteins, or the epitope peptides, would be very useful to facilitate targeted diagnosis and therapy. In recent years, the MIP-based synthetic receptors were extensively studied for specific tumor cell or tissue imaging owing to their unique properties, such as physical and chemical stability, ease of functionalization with fluorescence dye or quantum dots (QDs), adjustable formats, and size to suit the imaging applications [34]. In the initial stages, celltargeting MIPs were designed using the whole cells as the templates but were limited in preparation methods and applications, owing to their fragility, dramatically varying shape, and size during the imprinting process [12]. Considering the special molecular expression of cancer cell membranes (e.g., proteins and glycans) [35], cancer cell
targeting MIPs were then designed by imprinting and recognition of specific overexpressed glycans or proteins on the cancer cell surface. As is known, cell membrane glycans like glucuronic acid (GlcA), sialic acid (SA), fucose (Fuc), and mannose (Man) are commonly expressed on different cell lines [35]. Among them, SA has been the most widely employed as a general cancer biomarker. Given this, Yin et al. reported SA-imprinted NPs as surface-enhanced Raman scattering (SERS) nanotags for targeting cancer cell and tissue imaging (Fig. 5
7) [36]. In their work, p-aminothiophenolfunctionalized silver NPs were used as the Raman reporting core and modified by 4-formylphenylboronic acid. The imprinting process was carried out on the boronate affinity-oriented surface due to strong and dynamic interactions between phenylboronic acid and the cis-diol groups of SA [37,38]. The principle for selective cell imaging is illustrated in Fig. 5
7A and B. The introduction of molecularly imprinted synthetic receptors endowed SERS tags with high specificity toward the cancer biomarker of SA. When used as model cells for imaging, human hepatoma carcinoma cells (HepG-2) exhibited strong SERS signals after incubated with SAimprinted SERS tags, while normal hepatic cells (L-02) generated much weaker signals. The authors further applied the SERS nanotags to the imaging of cancer liver tissues; while cancer liver tissues exhibited apparent SERS signals, normal liver tissue showed little signal, demonstrating that SA-imprinted SERS nanotags were able to visualize cancer cells and

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FIGURE 5–7 (A) Schematic illustration of the preparation of Ag@SiO2 SERS tags and SA-imprinted on Ag@SiO2 SERS tags. (B) Schematic of the principle for SERS imaging of cancer cells and tissues via SA-imprinted nanotags. (C
H) SERS imaging of cells or liver tissues. Columns from left to right: bright-field, SERS image and representative SERS spectra at three locations on tissue surfaces as indicated by the symbol. (C) HepG-2 cells after incubation with boronic acid-functionalized NPs. (D) L-02 cells after incubation with boronic acid-functionalized NPs. (E) Cancer liver tissue after incubation with SA-imprinted SERS nanotags. (F) Normal liver tissue after incubation with SA-imprinted SERS nanotags. (G) Cancer liver tissue after incubation with nonimprinted SERS nanotags. (H) Normal liver tissue after incubation with nonimprinted SERS nanotags. NP, Nanoparticle; SA, sialic acid; SERS, surface-enhanced Raman scattering. Reproduced with permission from 2015 Royal Society of Chemistry.

tissues. Moreover, this study indicated that synthetic receptors with affinity toward specific tumor biomarkers could be combined with certain imaging techniques for rapid cancer screening and cancer-related studies. Another study on the SA-imprinted synthetic receptors for cell imaging was reported by Shinde et al. using a core
shell NP equipped with nitrobenzoxadiazole (NBD) fluorescent reporter groups (Fig. 5
8) [39]. The imprinting process was achieved by exploiting a hybrid approach combining reversible interactions between p-vinyl phenylboronic acid and SA,

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FIGURE 5–8 (A) Procedure for preparation of SA-MIPs. (B) Principle of using MIPs for imaging of SA terminated glycan motifs. (C and D) Optical microscope image of SA-MIPs. (E
H) Quantification of the expression level of the SA glycans with flow cytometry using SA-MIP probe or FITC-lectin. (I, J) Fluorescence microscopy images of DU 145 cells incubated with SA-MIP (I) and FITC-lectin (J) after nuclear staining using DAPI. FITC, Fluorescein isothiocyanate; MIP, molecularly imprinted polymer; SA, sialic acid. Reproduced with permission from 2015 American Chemical Society.

the introduction of cationic amine functionalities, and the use of an NBD-appended ureamonomer as a binary hydrogen-bond donor targeting the carboxylic acid and OH groups of SA molecules. The resultant SA-imprinted synthetic receptors exhibited good affinity to SA (K 5 6.6 3 105 M21 in 2% water, 5.9 3 103 M21 in 98% water), whereas binding of the competitor GlcA and other monosaccharides was considerably weaker (K 5 1.8 3 103 M21 in 98% water). In the cell imaging experiments, the SA-MIPs could selectively stain different cell lines in correlation with the SA expression level on cell membranes. Further study found that the SA-MIPNPs could selectively stain prostate cancer cell lines in a similar fashion as corresponding SA specific lectins. This study suggested that the application of the MIPs-based synthetic receptors for targeted glycomics and subsequently imaging cells would be reliable. With the same concept, reprecipitation was performed to prepare SA-imprinted fluorescent conjugated NPs for targeted cancer cell imaging [40]. In their cell imaging assay, DU 145 and HeLa cell lines with different SA glycan expression levels were chosen. The result again demonstrated that the SA-imprinted NPs could selectively bind with the SA overexpressed in DU 145 cancer cells. However, other auxiliary techniques or tools are required for further discrimination of different SA overexpressed cells. Different from imprinting SA for cancer cells, Haupt’s group recently demonstrated the applicability of the fluorescently labeled glycan-based nano-MIPs for normal cell and even tissue imaging [34,41]. GlcA was used as the template due to its abundance on keratinocyte surface, in the form of hyaluronan as a part of glycocalix [42]. The nano-MIPs are stable without any aggregation in the cell culture media, and the cell morphology was not

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influenced by incubation with the MIPNPs. Furthermore, the nano-MIPs were applied to human skin specimens to establish their usefulness for tissue imaging. The nano-MIPs bound to the skin tissue are mainly localized in the basal layer of the epidermis and the papillary dermis, while lower amounts of MIPs can be found in the cornified, granular, and spinous cell layer. This is consistent with the results obtained with FITC-labeled hyaluronic acid binding protein applied to tissue samples from the same batch and prepared in the same way. The same group also employed MIP-coated QDs (MIP-QDs) for imaging different cell membrane glycans, GlcA or N-acetylneuraminic acid (NANA) (Fig. 5
9) [43]. QDs are semiconductor nanocrystals with unique optical and electronic properties, such as size-tunable

FIGURE 5–9 (A) Polymeric shell was synthesized on the surface of InP/ZnS quantum dots. (B) A second shell of MIP is synthesized on the surface of the polymeric shell. (C) Binding affinity of MIPGlcA-QDs (black) and NIPGlcA-QDs (white) with [14C]glucuronic acid in water. (D) Relative fluorescence intensity of keratinocytes after imaging with MIP-QDs (black) and NIP-QDs (white). (E, F) Staining of keratinocytes (E) and KU812 cells (F) with MIP-QDs (green). (G) Binding affinity of MIPNANA-QDs (black) and NIPNANA-QDs (white) with [3H]sialic acid in water. (H) Relative fluorescence intensity of keratinocytes after imaging with MIP-QDs (black) and NIP-QDs (white). (I, J) Staining of keratinocytes (I) and KU812 (J) with MIP-QDs (red). (K) Confocal microscope image of staining the GlcA and NANA on human keratinocytes by MIPGlcA-QDs (green) and MIPNANA-QDs (red). MIP, Molecularly imprinted polymer; QDs, quantum dots. Reproduced with permission from 2016 Wiley-VCH.

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light emission, broad excitation, and narrow-emission spectra. They employed green- and red-emitting InP/ZnS QDs, which are capable of emitting green (550 nm) or red (660 nm) fluorescence under a UV lamp. Thus the authors considered the use of MIP-coated QDs with different visible light emissions and different affinities to cell membrane glycans for multiplexed targeting and imaging human keratinocytes. The recognition properties of MIP-QDs were evaluated by equilibrium radio ligand-binding assays. The results showed that MIP-QDs bound much more targeting molecules (i.e., GlcA or NANA) than the nonimprinted controls (NIP-QDs), indicating the creation of imprinted sites. In quantitative cell imaging experiments, MIP-QDs showed 48% more binding than NIP-QDs. Furthermore, staining nucleus with organic dyes proves that MIP-QDs can be coupled with other staining methods without a loss of specificity, which demonstrated the potential of MIP-based synthetic receptors as versatile multiplexed imaging tools when conjugated to QDs of different emission colors. The application of MIP-coated QDs as artificial receptors and imaging agents for glycosylation sites could pave the way for new applications in diagnostics, theranostics, and therapeutics. Exploitation of glycan-targeted synthetic receptors has been receiving a great deal of effort to further confirm that monosaccharide-imprinting MIPs can be used as a toolbox for specific cancer cell recognition. For example, Fuc and Man, another two glycans, were also imprinted on fluorescein isothiocyanate (FITC)-doped silica NPs via the boronate affinityoriented surface molecular imprinting strategy, and then were used for cancer cell imaging [44]. The obtained monosaccharide-imprinted FITC-modified NPs (MIP-FITC-NPs) were also verified to be able to differentiate cancer cells from normal cells. In their study, SA, Fuc, and Man-imprinted FITC-NPs were then used as fluorescent probes for cancerous cell imaging (HepG-2 and MCF-7 cell lines) with their noncancerous counterparts (L-02 and MCF-7 cell lines) as controls. The results showed that HepG-2 cells exhibited strong fluorescence, while L-02 cells showed few weak discrete fluorescent dots; MCF-7 cells exhibited weak fluorescence, while MCF-10A cells showed no fluorescence. These results clearly indicate that MIP-FITC-NPs permitted selective imaging of cancer cells from normal cells. Moreover, the fluorescence intensity in the images reflected the monosaccharide expression levels on different cancer cell types. For example, HepG-2 expresses comparable levels of SA, Fuc, and Man, while MCF-7 expresses lower levels of Fuc and Man than that of SA. This study confirmed that monosaccharide-imprinted synthetic receptors can be used as a general toolbox for specific recognition and visualization of cancer cells. Furthermore, MIP-based receptors with glycan affinity show great promise in many specific bio-applications; they can be applied to tissue imaging for pathological investigation. Also, they can be extended to other response mechanisms; monosaccharide-imprinted plasmonic NPs can be used for targeted photothermal therapy (discussed below) [45]. Recently, researchers in the field of cancer cell imaging have been consciously shifting their attention to other tumor-related biomarkers. Evidence suggested that vascular endothelial growth factor (VEGF) is overexpressed in various cancers cells, such as gastrointestinal, breast, and colorectal [46,47]. Therefore specific binding VEGF by artificial antibodies has been proposed to facilitate targeting tumor cell imaging. Cecchini et al. prepared human VEGF (hVEGF) imprinted polymers by a solid-phase synthesis strategy and coupled with QDs to enable subsequently fluorescent imaging in vivo (Fig. 5
10) [48]. In this study, to

FIGURE 5–10 (A) Synthesis of anti-hVEGF MIPs and the homing profile of anti-hVEGR QD-MIPs. (B) TEM images of QDs and QD-MIPs. (C) Scheme of the in vivo experiments. (D) Statistical analysis of the mean nanoprobe
cell distances in the three different scenarios. (E) Panel of the bright-field and fluorescence images of human melanoma cells, the fluorescent nanoprobes, and the overlay of the two signals. QD, quantum dot; MIP, molecularly imprinted polymer. Reproduced with permission from 2017 American Chemical Society.

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obtain a synthetic receptor with specific affinity toward the whole VEGF proteins, a synthetic nonapeptide epitope of hVEGF was used as the template during the imprinting process. As the authors declared, the resultant QD-coupled, molecularly imprinted, nano-sized synthetic receptors (QD-MIPs) offered significant advantages over antibodies, in relation to stability, efficiency, cost, and control over functionalization. SPR results indicated that the QD-MIPs with negligible toxicity had high affinity and specificity to both the epitope and the whole hVEGF protein in vitro. A melanoma cell line WM-266 known to overexpress hVEGF was exploited to create tumor xenografts in zebrafish embryos and used as the hVEGF(1) in vivo model [49]. In contrast, A-375 cell line, with low expression of hVEGF, was used as the hVEGF(2) model. The QD-MIPs or QD-NIPs (nonimprinted controls) were injected into the yolks of 48 hpf zebrafish embryos. After7 h of incubation, confocal microscopy was used to detect the in vivo distribution of injected nanoprobes and to compare it with the tumor mass localization. Notably, Fig. 5
10B shows that QD-MIPs were actually able to home toward the cells in the hVEGF(1) model and specifically localize in close proximity to the tumor mass. These findings again indicated the significant potential of MIP-based synthetic receptors for targeted cell imaging and site-specific NP homing, paving the way for additional studies to target other secreted factors in several human disorders. MIP-based synthetic receptors have raised interest in bio-imaging. In contrast to the other chemical imaging approaches, MIP-based synthetic receptors are one of the most successful and versatile tools in regards to selectivity. Furthermore, MIP-based synthetic receptors are cheaper and more robust than natural receptor probes. Finally, the flexibility in the selection of targeted molecules leads them to be promising candidates for the visualization of various tissues and cells. However, current solutions to the drawbacks of MIP-based synthetic receptors are insufficient, such as rapid photo-bleaching, potentially toxicity, and a relatively low targeting efficiency in vivo.

5.2.4 Cancer Therapy Inspired by the universal applications of MIPs in the field of tumor cell and tissue imaging, researchers have tried to imprint tumor biomarkers on NPs that facilitate targeted cancer therapy [50]. With the knowledge that a wide spread of transmembrane helices can serve as binding sites for corresponding membrane receptors recognition, Zhang et al. combined the scaffold-based peptide design with surface imprinting to fabricate a peptide-imprinted NP with high affinity to a cell membrane protein (Fig. 5
11) [51]. The template being used was a hybrid apamin-p32 polypeptide; protein p32 was overexpressed on the surface of various tumor cells and capable of mediating targeted drug delivery to tumor sites [52]. The authors used the fluorescence polarization technique to quantify the binding properties of MIPNPs with recombinant p32. The results of direct titration and competitive binding assays demonstrated that MIPNPs bind strongly to p32 protein (Fig. 5
11). Epitope-imprinted synthetic receptors demonstrated their targeting ability through a higher uptake of imprinted NPs than control NPs by p32-positive cancer cells. An in vivo study showed that the MIPNPs

FIGURE 5–11 (A) Preparation of MIPs for specific membrane protein recognition. (B) Amino acid sequence of p32, apamin, and template peptide. (C) Binding affinity of MIPNPs and NIPNPs to p32. (D) Competitive binding assays. In vivo distribution of nanoparticles in 4T1-tumor-bearing mice. (E) The 4T1tumor-bearing KM mice were intravenously injected with various nanoparticles. (F) The organs and tumors were examined after being injected with NPs for 24 h. (G) Semiquantitative results obtained using fluorescence imaging. (H) In vivo antitumor effect of PDT performed using different NPs. (I) Images of mice treated with NIPNPs (1) and MIPNPs (2). MIP, Molecularly imprinted polymer; NP, nanoparticle. Reproduced with permission from 2015 Wiley-VCH.

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encapsulating a fluorophore dye (methylene blue) led to a considerably higher accumulation of imprinted than of nonimprinted NPs in a mouse xenograft tumor, implying the potential to mediate targeted drug delivery for therapy. As expected, photodynamic treatment (PDT) showed that MB-encapsulating MIPNPs are more potent than nonimprinted NPs to inhibit cancer in 4T1 subcutaneous xenografts, indicating the great potential of a tailor-made synthetic receptor for selective cell targeting and its applications in cancer inhibition. To further investigate maintaining the stable conformation of the peptide template during preparation, the same group prepared a novel imprinted polymeric NP that could recognize the transmembrane domain of target receptors [53]. In their study, they used a transmembrane helical peptide as the template, and added 2,2,2-trifluoroethanol to maintain a stable conformation for the peptide template. Through imprinting the transmembrane domain of cell membrane receptors, the authors found that the MIPNPs showed a high internalization rate in vitro, and subsequently enhanced cellular uptake and permeability in target tissues for tumor-targeted drug delivery. This “hidden” epitope-imprinting method might be a novel tool to design artificial receptors for targeting tumor therapy. Liu’s group has made a pioneering effort to develop a glycan-imprinted gold nanorod (AuNR) for targeted cancer imaging and photothermal therapy [45]. They used AuNR as the core plasmonic nanomaterial and SA, which is overexpressed on the surface of most cancer cells, as the glycan template for the preparation of MIPs. After optimizing the imprinting process, the resultants, SA-imprinted AuNRs, exhibited excellent specificity and high affinity to SA molecules in vitro. Also, they exhibited excellent photothermal property; the solution temperature of SA-imprinted AuNRs could increase from 23 C to about 60 C after 6 min of laser irradiation at 1 W/cm2. Then, the SA-imprinted AuNRs were used for in vivo photothermal therapy on HepG-2 (human hepatoma carcinoma cells) tumor-bearing mice. The result showed that the SA-imprinted AuNR exhibited good cancer cell
targeting selectivity as well as high photothermal effect. Moreover, the targeted plasmonic nanomaterial was able to selectively kill tumor cells without damaging the surrounding healthy tissue. In short, using MIP-based synthetic receptors as drug-delivery vehicles for targeted cancer therapy is advantageous due to their flexible selectivity of targeting molecules and treatment methods; they can target not only the exposed transmembrane proteins or monosaccharide on the tumor cell membranes, but also the partially exposed transmembrane proteins via specific threedimensional recognition. Moreover, they can be flexibly used for targeting cell imaging, chemotherapy, and photothermal properties by using different MIP matrices. Although great progress in MIP-based synthetic receptors has been made for cell targeting, their biomedical applications from in vitro to in vivo still remain unclear. To efficiently deliver therapeutic agents to targeted cells or tissues in vivo, sometimes cumbersome chemistry is still required to achieve longevity in the bloodstream, which will open up new access to the design and preparation of cancer-specific nanomaterials for targeted photothermal therapy of cancer. To this end, Takeuchi et al. employed the albumin-imprinting strategy to regulate surface protein corona formation on MIPs, allowing the MIP nanogels (MIP-NGs, B50 nm) to hide in the bloodstream (Fig. 5
12) [54]. Previous results suggested that controlling protein corona components may affect the biological activity of NPs in vivo, for example, reducing nonspecific

FIGURE 5–12 (A) Preparation of MIP-NGs for HAS by emulsifier-free precipitation polymerization. (B) Concept of MIP-NGs acquires stealth in situ by cloaking with albumin. (C) Binding affinity of MIP-NGs and NIP-NGs to HSA. (D) Selectivity of MIP-NGs toward HSA, IgG, and Fib. (E) In vivo confocal fluorescence microscopy images of MIP-NGs and NIP-NGs retained in the liver. (F) Relative fluorescence intensities of MIP-NGs and NIP-NGs in vessels and liver cells. (G) In vivo confocal fluorescence microscopy images. (H) Fluorescence intensity of MIP-NGs in the HeLa tumor region and the normal region. MIP, Molecularly imprinted polymer; NG, nanogels. Reproduced with permission from 2017 Wiley-VCH.

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cellular uptake [55]. Therefore they chose the most abundant protein in blood (albumin) for preparing MIP-NGs to form an albumin-rich protein corona. Due to their high affinity and selectivity toward albumin, the MIP-NGs could acquire stealth in situ by cloaking with the body’s albumin after being administered in vivo. The excellent binding behavior of MIP-NGs was examined by injecting them onto a HAS-immobilized SPR sensor. Then the MIP nanogels were injected into the tail vein of a mouse to in vivo image their circulating in the blood vessels. Intravital fluorescence resonance energy transfer imaging of rhodamine-labeled albumin and fluorescein-conjugated MIP-NGs showed that albumin protein in blood could be captured by the MIP-NGs immediately after intravenous administration, forming an albumin-rich protein corona. The MIP nanogels could circulate in blood longer than nonalbumin-imprinted nanogels, even with almost no retention in liver tissue. It is worth mentioning that no pretreatment with albumin was necessary before MIP-NG infusion, preventing potential side reactions caused by exogenous proteins and simplifying the quality control of MIP-NGs during the manufacturing process. In addition, they found that the MIP-NGs passively accumulated in tumor tissue through enhanced permeability and retention effects, similar to the passive targeting of PEGylation [56]. This finding suggests that the molecular imprinted synthetic receptors, based on regulation of the selective protein corona in vivo, may significantly influence the development of drug nanocarriers for cancer theranostics.

5.2.5 Cell Isolation Dynamic interactions between cell membranes and the ECM are crucial to almost all kinds of cellular processes. For example, reversible cell adhesion behaviors resulting from these dynamic receptor
ligand interactions are very important in both physiology and pathology [57]. Recently, selective cell isolation via dynamic cell adhesion control on stimuli-responsive substrates has shown great promise in tissue engineering, drug targeting, and cell-based diagnostics [58
60]. Current strategies to achieve controlled cell adhesion mainly rely on the reversible ligand functionalization through reversible linkers (e.g., noncovalent or reversible covalent interactions). However, only a few reversible linkage chemistries have been successfully exploited. Considering the biomimetic and dynamic nature of synthetic receptors, they have been used for bioactivity surface introduction, and selective and reversible cell binding. Synthetic receptors with dynamic bioaffinity could be easily designed to perform selective cell capture and release. In fact, the development of synthetic receptors in cell isolation is uneven; only the molecular imprinting strategy has been employed recently [12]. The groups of Giardelli and Kazuhiko first studied cell adhesion or proliferation behaviors on MIP substrates that targeted to fibronectin (FN), a cell-adhesive protein [61,62]. Although the MIPs were designed with bioaffinity, the FN recognition properties in these studies were relatively weak, compromising the end objectives of the study. More importantly, the systems lacked an efficient mechanism for reversible modulating cell
material interactions, for example, achieving the transformation of cell-adhesive behaviors by dynamic FN binding. To this end, Pan et al. reported a novel system based on the molecularly imprinted synthetic receptors

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for reversible cell culture. The system relied on a PNIPAm-based MIP hydrogel layer with thermo-responsive affinity toward a cell-adhesive peptide RGDS (Arg-Gly-Asp) by redoxinitiated polymerization [63]. In the design, molecular imprinting methodology was employed to create the molecular recognition sites for targeted RGDS peptide onto the thermo-responsive PNIPAm substrate, which was innovatively used as a highly efficient system for harvesting cell sheets. Compared with simple physical absorption and covalent immobilization approaches to biomolecular immobilization, the surface thermo-responsive molecular recognition sites for cell-adhesive peptides did not only promote cell adhesion during cell culture, but also facilitated cell detachment during cell sheet harvesting. It was the first successful sample to module cell
material interactions and isolate cells efficiently using a synthetic receptor. However, it is worth mentioning that the poor accessibility of the bound RGD peptides embedding in recognition sites markedly limited bioactivity presentation on the material interfaces. Taking one step further, the same group recently reported an epitope-imprinting process [64] to dynamically display bioactive ligands on the material interface and control reversible cell adhesion (Fig. 5
13) [65]. They employed a terminal short peptide sequence (epitope peptide) of an RGD-based long peptide as a template during the imprinting process, thus

FIGURE 5–13 (A) Generation of an EIB and dynamic cell adhesion. (B) Binding isotherms for the epitope peptide to EIB and NIB in PBS. (C) Selective binding of EIB and NIB toward different FITC-labeled peptides in PBS. (D) Micrographs of mouse 3T3 cells cultured for 3 h on NIB, NIB with RGD-based peptide, EIB, EIB with RGD-based peptide, and EIB with nonadhesive RGE-based peptide, respectively. (E) Time-dependent detachment of the adhered cells from RGD-bound EIB by incubation in α-MEM with free epitope peptide. EIB, Epitope-imprinted biointerface; NIB, nonimprinted biointerface. Reproduced with permission from 2017 Wiley-VCH.

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allowing the resultant MIPs to bind to the terminal epitope and free up the bioactive RGD sequence in the opposite end. To obtain high epitope binding affinity, benzamidine-bearing monomer and carboxyl-containing epitope peptide templates were chosen to improve imprinting efficiency, in view of the strong electrostatic interaction in the benzamidine
carboxylate complex. In this design, the epitope peptide could act as a reversible anchor of the RGD peptide, leaving the latter exposed for interacting with the cell surface integrin receptors. Isothermal adsorption experiments revealed that the epitope-imprinted biointerface (EIB) exhibited a higher association constant (Ka 5 9.75 3 107 M21) than that of the nonimprinted biointerface (Ka 5 0.81 3 107 M21). Moreover, the EIB had excellent selectivity toward the epitope peptide, further confirming the formation of specific recognition sites for the epitope peptide after the molecular imprinting process. Qualitative and quantitative analysis on the fluorescence binding experiments demonstrated that the EIB could stably bind to the epitope peptide through surface epitope recognition sites and release it via epitope molecular exchange. With dynamic EIB in hand, the authors further investigated the cell adhesion behaviors. Without binding RGD to the EIB, the cell morphology only showed a typical nonadhesive round shape. In contrast, cell adhesion behavior on EIB was dramatically changed if the surface was bound with RGD. Furthermore, when the initial cell culture medium α-MEM was changed to another α-MEM containing 0.1 mg/mL free epitope peptide, a gradual transition of cell morphology, from a spread-out shape to a round shape, could be clearly observed in the first 4.5 h. This result indicated that the molecularly imprinted dynamic biointerface could regulate cell adhesion behaviors from attachment to detachment, leading to great promise for cell isolation. In addition, this study highlights the importance of the dynamic nature of molecularly imprinted receptors. Compared to other strategies for dynamic biointerface fabrication, such as reversible covalent or host
guest chemistry, such a molecularly tunable dynamic system based on surface engineering of synthetic receptors may unlock new applications in in situ cell biology, diagnostics, and regenerative medicine. Another application of synthetic receptors for cell isolation is their potential in blood typing. Using an imprint process, Hayden et al. designed an erythrocyte-specific biosensor device on a QCM transducer surface for ABO blood grouping (Fig. 5
14) [66]. Although erythrocytes of different blood groups (A, B, AB, and O) are morphologically identical, their surface antigens are different. Hence, the selectivity of the sensor device is mainly reliant on the molecularly imprinted cavities (i.e., MIP-based synthetic receptors) with multiple noncovalent interactions between the sugar residue antigens on an erythrocyte membrane. The authors found that the MIP-based receptors on the device clearly discriminated the blood group used as an imprinting template, thus showing the possibility for blood-group typing. Recently, Piletsky et al. prepared blood-B-antigen-specific MIPs with magnetism for selective blood cell capture and typing [67]. It was found that the amount of blood type B bound to the B antigen-specific MIPs was much higher than that of blood type O, while fewer blood type B was bound to maltotriose-specific MIPs due to nonspecific binding. Followed by magnetically induced decolorization, this method enables visual detection of blood types.

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FIGURE 5–14 (A) Preparation of erythrocyte-imprinted polymer with soft lithography. (B) The imprint site of red blood cells on the polyurethane layer. (C) Imprint site of a single red blood cell. (D) The adsorption of red blood cells on imprinted or nonimprinted polyurethane. (E) Selectivity of the MIP layers for intact cells. (F) The selectivity of ABO-imprinted layers. (G) Blood-group typing with whole blood samples. MIP, molecularly imprinted polymer. Reproduced with permission from 2006 Wiley-VCH.

5.2.6 Other Potentials Besides the above established applications, synthetic receptors have been exploited for some other potential uses in biomedicines. Recently, the affinity screening strategy was used to design receptor-like NPs with high affinity of growth factors, such as VEGF. Evidence suggested that VEGF plays a major role in angiogenesis and is overexpressed in various cancers cells, such as gastrointestinal, breast, and colorectal [68]. Thus VEGF is a critical factor during the early stages of tumor growth [69]. Given this, Koide et al. prepared a polymer NP with engineered affinity for VEGF165 by incorporating monomers trisulfated N-acetylglucosamine (3,4,6S-GlcNAc) and N-tert-butylacrylamide (TBAm) in cross-linked PNIPAm copolymer NPs (Fig. 5
15) [70]. The authors found that the NP affinity can be “tuned” by varying the amount of both 3,4,6S-GlcNAc and TBAm monomer incorporation. Increasing the 3,4,6SGlcNAc monomer and/or decreasing the hydrophobic monomer (TBAm) content could decrease affinity to the protein. After optimization, the NP with high affinity toward VEGF165 was first applied for in vitro VEGF inhibition. As is known, binding of VEGF165 to VEGFR-2, its native receptor induces the phosphorylation of VEGFR-2 and triggers downstream cellsignaling events. As NP sequestered VEGF165, it strongly inhibits phosphorylation (Tyr 951) at a concentration of 10 μg/mL. Due to the angiogenesis of VEGF, the authors examined the antiangiogenic effect of the NP in vivo in Matrigel plug in living mice. As expected, the NP (30 μg/mL) significantly inhibited VEGF165-induced HUVEC migration and invasion, and

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FIGURE 5–15 (A) Chemical composition of the polymer NPs. (B) Effect of 3,4,6S-GlcNAc monomer amount on the binding affinity of the NPs. (C) TEM images of NP11. (D) In vitro VEGF-inhibition experiment and comparison with heparin. Antiangiogenic effect of NPs. (E) Inhibition of VEGF165-dependent cell motility by NP1 and NP11
NP13. (F) Inhibition of VEGF165-dependent capillary tube formation in the presence of NP1 and NP11
NP13. (G) Inhibition of in vivo angiogenesis in Matrigel plugs implanted in mice. NP, Nanoparticle; VEGF, vascular endothelial growth factor. Reproduced with permission from 2017 Nature.

subsequently capillary tube formation. The results verified that synthetic receptor NPs can be engineered to bind to and interfere with a signaling protein, implying their potential in regenerative medicine and even cancer therapy. In addition, the protein affinity of synthetic receptors has also been employed to control the functions or structures of target proteins. Nakamoto et al. reported the potential of protein-affinity receptor-like NPs for refolding denatured protein [71]. The affinity-screened polymeric NPs, prepared by copolymerizing optimized combinations and populations of functional monomers, were capable of facilitating resolubilization and refolding of aggregated lysozymes. The authors revealed that resolubilization and refolding aggregated lysozymes are driven by a strong affinity of NPs to denatured lysozymes, as well as a relatively weak affinity to native lysozymes. After centrifugal ultrafiltration, the refolded lysozyme showed native conformation and enzymatic activity. These results suggest the great potential of synthetic receptors as artificial chaperones with high facilitating activity for nature biomolecules. Another promising application of synthetic receptors with protein affinity is their ability to assist protein crystallization. Since MIP-based synthetic receptors contain complementary cavities that are capable of rebinding targeting protein; the fingerprint of the targeting

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protein on the polymer allows it to be an ideal template for crystal formation. Saridakis et al. demonstrated that MIPs indeed facilitate the formation of large single-protein crystals at metastable conditions [72]. This is due to the recognition of proteins by the cavities, which concentrate the target proteins near the interface and lead to protein crystallization. This initiative research implied that protein-affinity synthetic receptors could act as nucleationinducing substrates by harnessing the target proteins as templates, thus significantly accelerating the discovery of new protein crystal structures [73].

5.3 Conclusion and Perspective In summary, synthetic receptors with bioaffinity have been extensively explored for biorelated applications due to their excellent tolerance and tunable affinity toward targeted molecules. To date, many novel strategies have been initiated to target effective receptors that can selectively associate with specific guest molecules. However, synthetic receptors with biomolecular affinity are mainly confined to the molecular imprinting and affinity screening strategies. Molecular imprinting can endow polymeric receptors with specific recognition sites complementary to target bio-related molecules. It has been shown to be the most successful technique regarding binding efficiency and selectivity due to the “lock and key” mechanism, similar to natural receptor
ligand interactions. In fact, initial applications of MIP-based synthetic receptors mainly focused on the uses in adsorption, separation, sensing, catalysis, etc. Until the last decade, this kind of synthetic receptor was used for biological or medical applications, like toxin neutralization, bacteriostasis, cell imaging, inhibition, isolation and cell adhesion behavior regulation, promoting the development of MIP-based synthetic receptors with bioaffinity. In contrast, the affinity screening strategy is simpler and less attractive for the design of synthetic receptors, because it derives from random copolymerization via the regulation of chemical compositions and functional groups. Nevertheless, these receptor mimics still exhibit comparable potential in biomedicine. Likewise, affinityscreened receptors can be employed for toxin neutralization, protein refolding, as well as the inhibition of specific cell signaling that are promised in cancer therapy and regenerative medicine. One of the ultimate goals of synthetic receptors is to implement the specific bioaffinity into a range of novel bio-related applications. We have given a brief summary of recent diagnostic and therapeutic applications of synthetic receptors in exploring novel smart biomaterials, devices, and biofunctions of disease. The potentials of synthetic receptors are far beyond current development, and we believe they should be further explored. As there is an increase in the number of synthetic receptors discovered toward new targeted biomolecules, more emerging applications will be found and give rise to versatile advanced biomaterials. Combination of synthetic receptors with more sensitive sensor platforms and microfluidic devices is expected to yield more appealing commercial developments and applications. Moreover, incorporating high-throughput synthesis and new analysis techniques in molecular imprinting and affinity screening strategy, as well as combining the exploitation of novel

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functional monomers, are conductive to generating inexpensive and bio-selective receptorlike nanomaterials, and lead to unpredictable advances in biotechnology. However, the challenges faced in synthetic receptors for biomedicine are exponentially higher than their traditional applications. For example, current synthetic receptors can only interact with cell membrane molecules to modulate simple cell behaviors like cell adhesion and cell apoptosis, cell migration and differentiation still remain difficult to control. In addition, more effort should be made to improve the performance and implantation of synthetic receptors in vivo [54]. There is no doubt that synthetic receptors have many feasible applications, and we believe that the increasing attention on synthetic receptors with bioaffinity will be essential to bridging the gap between chemical science and biomedicine.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21574091 and 21875092) and the Natural Science Foundation of Jiangsu Province (BK20160056).

References [1] W.P. Daley, S.B. Peters, M. Larsen, Extracellular matrix dynamics in development and regenerative medicine, J. Cell Sci. 121 (3) (2008) 255
264. [2] N. Huebsch, D.J. Mooney, Inspiration and application in the evolution of biomaterials, Nature 462 (2009) 426. [3] S. Subrahmanyam, S.A. Piletsky, A.P. Turner, Application of natural receptors in sensors and assays, Anal. Chem. 74 (16) (2002) 3942
3951. [4] C.S. Mahon, D.A. Fulton, Mimicking nature with synthetic macromolecules capable of recognition, Nat. Chem. 6 (8) (2014) 665
672. [5] V.J.B. Ruigrok, M. Levisson, M.H.M. Eppink, H. Smidt, J. van der Oost, Alternative affinity tools: more attractive than antibodies? Biochem. J. 436 (1) (2011) 1
13. [6] G. Vlatakis, L.I. Andersson, R. Müller, K. Mosbach, Drug assay using antibody mimics made by molecular imprinting, Nature 361 (6413) (1993) 645
647. [7] D. Li, Y. Chen, Z. Liu, Boronate affinity materials for separation and molecular recognition: structure, properties and applications, Chem. Soc. Rev. 44 (22) (2015) 8097
8123. [8] K. Ariga, H. Ito, J.P. Hill, H. Tsukube, Molecular recognition: from solution science to nano/materials technology, Chem. Soc. Rev. 41 (17) (2012) 5800
5835. [9] J. Hu, S. Liu, Engineering responsive polymer building blocks with host
guest molecular recognition for functional applications, Acc. Chem. Res. 47 (7) (2014) 2084
2095. [10] A. Harada, Y. Takashima, M. Nakahata, Supramolecular polymeric materials via cyclodextrin
guest interactions, Acc. Chem. Res. 47 (7) (2014) 2128
2140. [11] W. Chen, Y. Ma, J. Pan, Z. Meng, G. Pan, B. Sellergren, Molecularly imprinted polymers with stimuliresponsive affinity: progress and perspectives, Polymers 7 (9) (2015) 1478. [12] J. Pan, W. Chen, Y. Ma, G. Pan, Molecularly imprinted polymers as receptor mimics for selective cell recognition, Chem. Soc. Rev. 47 (15) (2018) 5574
5587.

Chapter 5 • Synthetic Receptors With Bioaffinity for Biomedical Applications

139

[13] J. O’Brien, K.J. Shea, Tuning the protein corona of hydrogel nanoparticles: the synthesis of abiotic protein and peptide affinity reagents, Acc. Chem. Res. 49 (6) (2016) 1200
1210. [14] J.P. Chippaux, M. Goyffon, Venoms, antivenoms and immunotherapy, Toxicon 36 (6) (1998) 823
846. [15] C. Maya Devi, M. Vasantha Bai, A. Vijayan Lal, P.R. Umashankar, L.K. Krishnan, An improved method for isolation of anti-viper venom antibodies from chicken egg yolk, J. Biochem. Biophys. Methods 51 (2) (2002) 129
138. [16] A. Weisman, B. Chou, J. O’Brien, K.J. Shea, Polymer antidotes for toxin sequestration, Adv. Drug Deliv. Rev. 90 (2015) 81
100. [17] Y. Hoshino, W.W. Haberaecker, T. Kodama, Z. Zeng, Y. Okahata, K.J. Shea, Affinity purification of multifunctional polymer nanoparticles, J. Am. Chem. Soc. 132 (39) (2010) 13648
13650. [18] Y. Hoshino, H. Koide, K. Furuya, W.W. Haberaecker, S.-H. Lee, T. Kodama, et al., The rational design of a synthetic polymer nanoparticle that neutralizes a toxic peptide in vivo, Proc. Natl. Acad. Sci. U.S.A. 109 (1) (2012) 33
38. [19] A. Weisman, Y.A. Chen, Y. Hoshino, H. Zhang, K. Shea, Engineering nanoparticle antitoxins utilizing aromatic interactions, Biomacromolecules 15 (9) (2014) 3290
3295. [20] J. O’Brien, S.-H. Lee, S. Onogi, K.J. Shea, Engineering the protein corona of a synthetic polymer nanoparticle for broad-spectrum sequestration and neutralization of venomous biomacromolecules, J. Am. Chem. Soc. 138 (51) (2016) 16604
16607. [21] Y. Wada, H. Lee, Y. Hoshino, S. Kotani, K.J. Shea, Y. Miura, Design of multi-functional linear polymers that capture and neutralize a toxic peptide: a comparison with cross-linked nanoparticles, J. Mater. Chem. B 3 (8) (2015) 1706
1711. [22] H. Lee, Y. Hoshino, Y. Wada, Y. Arata, A. Maruyama, Y. Miura, Minimization of synthetic polymer ligands for specific recognition and neutralization of a toxic peptide, J. Am. Chem. Soc. 137 (34) (2015) 10878
10881. [23] Y. Hoshino, T. Kodama, Y. Okahata, K. Shea, Peptide imprinted polymer nanoparticles: a plastic antibody, J. Am. Chem. Soc. 130 (46) (2008) 15242
15243. [24] Y. Hoshino, H. Koide, T. Urakami, H. Kanazawa, T. Kodama, N. Oku, et al., Recognition, neutralization, and clearance of target peptides in the bloodstream of living mice by molecularly imprinted polymer nanoparticles: a plastic antibody, J. Am. Chem. Soc. 132 (19) (2010) 6644
6645. [25] L. Wen, D. Kai, R. Jinsong, Q. Xiaogang, A β-lactamase-imprinted responsive hydrogel for the treatment of antibiotic-resistant bacteria, Angew. Chem. 128 (28) (2016) 8181
8185. [26] J. Zhang, Y.P. Chen, K.P. Miller, M.S. Ganewatta, M. Bam, Y. Yan, et al., Antimicrobial metallopolymers and their bioconjugates with conventional antibiotics against multidrug-resistant bacteria, J. Am. Chem. Soc. 136 (13) (2014) 4873
4876. [27] A. Motib, A. Guerreiro, F. Al-Bayati, E. Piletska, I. Manzoor, S. Shafeeq, et al., Modulation of quorum sensing in a gram-positive pathogen by linear molecularly imprinted polymers with anti-infective properties, Angew. Chem. 129 (52) (2017) 16782
16785. [28] M.B. Miller, B.L. Bassler, Quorum sensing in bacteria, Annu. Rev. Microbiol. 55 (1) (2001) 165
199. [29] H.F. Kahya, P.W. Andrew, H. Yesilkaya, Deacetylation of sialic acid by esterases potentiates pneumococcal neuraminidase activity for mucin utilization, colonization and virulence, PLoS Pathog. 13 (3) (2017) e1006263. [30] L. Ma, S. Feng, C. Fuente-Núñez, R.E.W. Hancock, X. Lu, Development of molecularly imprinted polymers to block quorum sensing and inhibit bacterial biofilm formation, ACS Appl. Mater. Interfaces 10 (22) (2018) 18450
18457. [31] G.F. Kaufmann, R. Sartorio, S.-H. Lee, J.M. Mee, L.J. Altobell, D.P. Kujawa, et al., Antibody interference with N-acyl homoserine lactone-mediated bacterial quorum sensing, J. Am. Chem. Soc. 128 (9) (2006) 2802
2803.

140

THERANOSTIC BIONANOMATERIALS

[32] D.J. Stephens, V.J. Allan, Light microscopy techniques for live cell imaging, Science 300 (5616) (2003) 82
86. [33] J.A. Barreto, O.M. William, K. Manja, G. Bim, S. Holger, S. Leone, Nanomaterials: applications in cancer imaging and therapy, Adv. Mater. 23 (12) (2011) H18
H40. [34] M. Panagiotopoulou, S. Kunath, K. Haupt, B. Tse Sum Bui, Cell and tissue imaging with molecularly imprinted polymers, in: T. Tiller (Ed.), Synthetic Antibodies: Methods and Protocols, Springer New York, New York, 2017, pp. 399
415. [35] E. RodrÍguez, S.T.T. Schetters, Y. van Kooyk, The tumour glyco-code as a novel immune checkpoint for immunotherapy, Nat. Rev. Immunol. 18 (2018) 204. [36] D. Yin, S. Wang, Y. He, J. Liu, M. Zhou, J. Ouyang, et al., Surface-enhanced Raman scattering imaging of cancer cells and tissues via sialic acid-imprinted nanotags, Chem. Commun. 51 (100) (2015) 17696
17699. [37] X. Wu, Z. Li, X.-X. Chen, J.S. Fossey, T.D. James, Y.-B. Jiang, Selective sensing of saccharides using simple boronic acids and their aggregates, Chem. Soc. Rev. 42 (20) (2013) 8032
8048. [38] G. Pan, B. Guo, Y. Ma, W. Cui, F. He, B. Li, et al., Dynamic introduction of cell adhesive factor via reversible multicovalent phenylboronic acid/cis-diol polymeric complexes, J. Am. Chem. Soc. 136 (17) (2014) 6203
6206. [39] S. Shinde, Z. El-Schich, A. Malakpour, W. Wan, N. Dizeyi, R. Mohammadi, et al., Sialic acid-imprinted fluorescent core
shell particles for selective labeling of cell surface glycans, J. Am. Chem. Soc. 137 (43) (2015) 13908
13912. [40] R. Liu, Q. Cui, C. Wang, X. Wang, Y. Yang, L. Li, Preparation of sialic acid-imprinted fluorescent conjugated nanoparticles and their application for targeted cancer cell imaging, ACS Appl. Mater. Inter. 9 (3) (2017) 3006
3015. [41] K. Stephanie, P. Maria, M. Jacqueline, M. Nataliya, S. Jörg, H. Karsten, Cell and tissue imaging with molecularly imprinted polymers as plastic antibody mimics, Adv. Healthc. Mater 4 (9) (2015) 1322
1326. [42] J. Turnbull, A. Powell, S. Guimond, Heparan sulfate: decoding a dynamic multifunctional cell regulator, Trends Cell Biol. 11 (2) (2001) 75
82. [43] P. Maria, S. Yolanda, B. Selim, K. Stephanie, D. Luminita, P. Elise, et al., Molecularly imprinted polymer coated quantum dots for multiplexed cell targeting and imaging, Angew. Chem. Int. Ed. 55 (29) (2016) 8244
8248. [44] S. Wang, D. Yin, W. Wang, X. Shen, J.-J. Zhu, H.-Y. Chen, et al., Targeting and imaging of cancer cells via monosaccharide-imprinted fluorescent nanoparticles, Sci. Rep. 6 (2016) 22757. [45] D. Yin, X. Li, Y. Ma, Z. Liu, Targeted cancer imaging and photothermal therapy via monosaccharideimprinted gold nanorods, Chem. Commun. 53 (50) (2017) 6716
6719. [46] C. Lange, E. Storkebaum, C.R. de Almodóvar, M. Dewerchin, P. Carmeliet, Vascular endothelial growth factor: a neurovascular target in neurological diseases, Nat. Rev. Neurol. 12 (2016) 439. [47] N. Ferrara, A.P. Adamis, Ten years of anti-vascular endothelial growth factor therapy, Nat. Rev. Drug Discov. 15 (2016) 385. [48] A. Cecchini, V. Raffa, F. Canfarotta, G. Signore, S. Piletsky, M.P. MacDonald, et al., In vivo recognition of human vascular endothelial growth factor by molecularly imprinted polymers, Nano Lett. 17 (4) (2017) 2307
2312. [49] L. Yang, G. Chen, S. Mohanty, G. Scott, F. Fazal, A. Rahman, et al., GPR56 regulates VEGF production and angiogenesis during melanoma progression, Cancer Res. 71 (2011) 5558
5568. [50] D. Peer, J.M. Karp, S. Hong, O.C. Farokhzad, R. Margalit, R. Langer, Nanocarriers as an emerging platform for cancer therapy, Nat. Nanotechnol. 2 (2007) 751.

Chapter 5 • Synthetic Receptors With Bioaffinity for Biomedical Applications

141

[51] Y. Zhang, C. Deng, S. Liu, J. Wu, Z. Chen, C. Li, W. Lu, et al., Active targeting of tumors through conformational epitope imprinting, Angew. Chem. 127 (17) (2015) 5246
5249. [52] V. Fogal, L. Zhang, S. Krajewski, E. Ruoslahti, Mitochondrial/cell-surface protein p32/gC1qR as a molecular target in tumor cells and tumor stroma, Cancer Res. 68 (17) (2008) 7210
7218. [53] S. Peng, Y. Wang, N. Li, C. Li, Enhanced cellular uptake and tumor penetration of nanoparticles by imprinting the “hidden” part of membrane receptors for targeted drug delivery, Chem. Commun. 53 (81) (2017) 11114
11117. [54] T. Toshifumi, K. Yukiya, S. Reo, Y. Takuya, T. Kazuko, M. Yu, et al., Molecularly imprinted nanogels acquire stealth in situ by cloaking themselves with native dysopsonic proteins, Angew. Chem. 129 (25) (2017) 7194
7198. [55] S. Susanne, L. Katharina, M. Volker, Controlling the stealth effect of nanocarriers through understanding the protein corona, Angew. Chem. Int. Ed. 55 (31) (2016) 8806
8815. [56] F.M. Veronese, G. Pasut, PEGylation, successful approach to drug delivery, Drug Discov. Today 10 (21) (2005) 1451
1458. [57] L. Liu, X. Tian, Y. Ma, Y. Duan, X. Zhao, G. Pan, A versatile dynamic mussel-inspired biointerface: from specific cell behavior modulation to selective cell isolation, Angew. Chem. Int. Ed. 57 (26) (2018) 7878
7882. [58] G. Pan, S. Sun, W. Zhang, R. Zhao, W. Cui, F. He, et al., Biomimetic design of mussel-derived bioactive peptides for dual-functionalization of titanium-based biomaterials, J. Am. Chem. Soc. 138 (45) (2016) 15078
15086. [59] A.M. Rosales, K.S. Anseth, The design of reversible hydrogels to capture extracellular matrix dynamics, Nat. Rev. Mater. 1 (2016) 15012. [60] B. Guo, G. Pan, Q. Guo, C. Zhu, W. Cui, B. Li, et al., Saccharides and temperature dual-responsive hydrogel layers for harvesting cell sheets, Chem. Commun. 51 (4) (2015) 644
647. [61] G. Ciardelli, D. Silvestri, C. Cristallini, N. Barbani, P. Giusti, The relevance of the transfer of molecular information between natural and synthetic materials in the realisation of biomedical devices with enhanced properties, J. Biomater. Sci. Polym. Ed. 16 (2) (2005) 219
236. [62] K. Fukazawa, K. Ishihara, Fabrication of a cell-adhesive protein imprinting surface with an artificial cell membrane structure for cell capturing, Biosens. Bioelectron. 25 (3) (2009) 609
614. [63] G. Pan, Q. Guo, Y. Ma, H. Yang, B. Li, Thermo-responsive hydrogel layers imprinted with RGDS peptide: a system for harvesting cell sheets, Angew. Chem. Int. Ed. 52 (27) (2013) 6907
6911. [64] N. Hidekazu, H. Chin-Shiou, K.J. Shea, Selective protein capture by epitope imprinting, Angew. Chem. 118 (15) (2006) 2452
2456. [65] G. Pan, S. Shinde, S.Y. Yeung, M. Jakˇstait˙e, Q. Li, A.G. Wingren, et al., An epitope-imprinted biointerface with dynamic bioactivity for modulating cell
biomaterial interactions, Angew. Chem. Int. Ed. 56 (50) (2017) 15959
15963. [66] O. Hayden, K.J. Mann, S. Krassnig, F.L. Dickert, Biomimetic ABO blood-group typing, Angew. Chem. Int. Ed. 45 (16) (2006) 2626
2629. [67] S. Piletsky, S. Rabinowicz, Z. Yang, C. Zagar, E.V. Piletska, A. Guerreiro, et al., Development of molecularly imprinted polymers specific for blood antigens for application in antibody-free blood typing, Chem. Commun. 53 (11) (2017) 1793
1796. [68] D. Shweiki, A. Itin, D. Soffer, E. Keshet, Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis, Nature 359 (1992) 843. [69] D.J. Hicklin, L.M. Ellis, Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis, J. Clin. Oncol. 23 (5) (2005) 1011
1027.

142

THERANOSTIC BIONANOMATERIALS

[70] H. Koide, K. Yoshimatsu, Y. Hoshino, S.-H. Lee, A. Okajima, S. Ariizumi, et al., A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165), Nat. Chem. 9 (2017) 715. [71] M. Nakamoto, T. Nonaka, K.J. Shea, Y. Miura, Y. Hoshino, Design of synthetic polymer nanoparticles that facilitate resolubilization and refolding of aggregated positively charged lysozyme, J. Am. Chem. Soc. 138 (13) (2016) 4282
4285. [72] E. Saridakis, S. Khurshid, L. Govada, Q. Phan, D. Hawkins, G.V. Crichlow, et al., Protein crystallization facilitated by molecularly imprinted polymers, Proc. Natl. Acad. Sci. U.S.A. 108 (2011) 11081
11086. [73] M.J. Whitcombe, Smart hydrogel crystal gardens, Nat. Chem. 3 (2011) 657.

6 Calcium Phosphate NanoparticleBased Systems for Therapeutic Delivery Yun Piao , Ho Pan Bei , Allison Tam, Yuhe Yang, Qiang Zhang, Mo Yang, Xin Zhao DEPARTMENT OF BIOMEDICAL ENGINEERING, THE H ONG K ONG POLYTECHNIC UNIVERSITY, KOWLOON, HONG K ONG S AR, P.R. C HINA

6.1 Introduction Nanoparticles (NPs) have been widely used to deliver therapeutic reagents due to their tunable size and outstanding surface characteristics, which endow them with the ability to accumulate in cells and result in increased intracellular drug concentrations [1]. Over the years, both organic (represented by polymers) and inorganic [e.g., calcium phosphates (CaPs)] materials have been used to fabricate these NPs. Even though polymer-based drug carriers have dominated the clinical market for many years due to their grand bioavailability, better encapsulation and control release, and lower toxicity, they still suffer from several drawbacks including lower mechanical strength, poor biochemical stability, and biocompatibility, limiting their further applications [2,3]. Therefore it is of great significance to explore inorganic-based NPs and their potential to promote scientific progress in drugdelivery devices. Recent researches have indicated that inorganic materials like silica [4], iron oxides [5], gold [6], titanium dioxide [7], and CaPs [8] hold great potential in drug-delivery applications. Among these inorganic drug-delivery systems (DDSs), CaP-based nanomaterials are found to be the most promising, due to their superior biocompatibility and biodegradability [9]. Compared with the abovementioned inorganic materials, CaPs also offer many advantages including their simplicity in production, low cost, adsorption abilities, and biochemical stability [2,10]. Notably, CaPs can remain relatively stable at normal physiological conditions (pH 5 7.4), no matter what the Ca/P ratio, phase, or crystallinity changes, but they are prone to dissolution in acidic environments (pH , 6.5), such as within lysosomes or around solid tumors [9]. Such pH-dependent solubility endows CaPs’ responsiveness to pH capable of 

These authors contributed equally to this chapter.

Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00006-7 © 2019 Elsevier Inc. All rights reserved.

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smart drug release in vitro and in vivo. Moreover, CaPs could be internalized into cells after a few hours by endocytosis, and the loaded drugs or biomolecules would be transported directly into living systems with minimal side effects [11]. Furthermore, Ca21 and PO32 4 , the degradation products of CaPs, are intrinsic to the body and would not lead to an immunogenic response or pose a threat to the system [12]. All of these merits ensure CaPs’ success as drug-delivery devices. Up until now, different forms of CaP NPs, such as bare CaP NPs, core shell, single, or multilayer CaP NPs, and coated CaPs, have been extensively explored as DDSs for the prevention and/or treatment of cancer, osteoporosis, inflammation, and infectious diseases such as periimplantitis or osteomyelitis in recent years [2,10 12]. Bioactive molecules and/ or drugs can be adsorbed onto the surfaces of CaP NPs after the formation or encapsulation into the NPs during the preparation process, providing a shelter for the loaded cargoes from extracellular degradation or oxidation, and a sudden release profile [2]. Herein, we summarize the state of the art of CaP NPs used as DDSs. This chapter is organized into four sections beginning with an introduction into the advantages of CaP NPs as DDSs. In the second section, therapeutics delivered using CaP NPs are described, followed by the third section discussing various types of CaP NPs based on their structural differences and their applications for the treatment of diverse diseases. Finally, we give a summary and perspectives on using CaPs as the next generation of drug carriers in the fourth section.

6.2 Therapeutics Delivered Using Calcium Phosphate Nanoparticles 6.2.1 Type of Therapeutics Therapeutics are molecules that benefit the treatment or reduction of disease damage. To date there are mainly three types of therapeutics delivered by CaP NPs: small-molecule drugs, proteins, and nucleic acids [13]. Small-molecule drugs are substances with low molecular weight which can directly enter cells and affect the functional groups of other molecules such as proteins. For example, chemotherapeutic drugs like doxorubicin and gemcitabine are highly effective at treating cancers because they can halt DNA synthesis and further kill the daughter cells of residual cell replication [14]. However, due to the cytotoxicity of these drugs, there were side effects like the loss of hair, rashes, and fever when they were used [15,16]. Facing the challenges of these side effects, researchers found that the encapsulation of said drugs in CaP NPs can drastically slow down drug leakage and only release encapsulated drugs when taken up by cells [17,18]. Using CaP NPs as vehicles, the indiscriminate attack by chemotherapeutic drugs can be upgraded to targeted therapy on cancer cells. Proteins are biomacromolecules that have also been widely explored for disease treatment. In contrast to small-molecule drugs, proteins have difficulty passing through the plasma membrane, and some are relatively fragile and easily degradable by stomach acids [19]. CaP NPs, with their positive charges, can act as vehicles for these large molecules to be taken

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up by cells. For example, by encapsulation in CaP NPs, hormones like insulin can be taken orally and reach intestines while remaining intact for the targeted treatment of diabetes [20]. Nucleic acids like DNAs and RNAs are extensively used genetic molecules due to their potential to cure diseases from its genetic root, including inherited diseases, cancer, and chronic infections [21]. However, owing to the phosphate group’s innate negative charges, DNAs and RNAs cannot enter cells without a suitable carrier [22]. Devastatingly, DNAs and RNAs will lose their therapeutic effects when they are degraded during delivery. In this regard, CaP NPs have been considered as exceptionally suitable transfection agents due to their charged properties that allow for their strong affinity with DNA and RNA molecules, and the promotion of endocytic delivery to cells [22].

6.2.2 Intracellular Uptake and Release Mechanism With therapeutics safely encapsulated in CaP NPs, they must enter the cells and remain mostly intact to achieve the desired therapeutic effects. CaP NP-based drug releases are typically erosion-controlled and activated by intracellular uptake (Fig. 6 1) [24]. The intracellular delivery of CaP NPs is mainly through two endocytosis pathways: phagocytosis and pinocytosis [25]. Both endocytosis pathways result in the NP encapsulated within a vesicle inside the cell body called an endosome. The endosome is then fused with a lysosome, forming an endolysosome (pH 5.5) around the NPs and degrading the encapsulated CaP NPs. This dissolution generates a high concentration of calcium and hydrogen phosphate ions, leading to an influx of water by osmosis and causing the lysosome to rupture. Therapeutics, no longer encapsulated within the CaP NPs, are released into the cytosol while excess calcium is pumped out by active transport of the cell. Therapeutic molecules are now free to move within the cytoplasm by diffusion and reach their target locations. Regarding the delivery of DNA molecules, their transfer into the nucleus by diffusion or dissociation through the nuclear pores is required, where they are incorporated into replicating strands where needed [21]. DNA-integrated strands will alter the transfected cells’ activities, producing the proteins required to release therapeutics on the targeted area. In contrast, RNA-integrated strands remain in the cytosol, where the transfected RNA will recognize and destroy the specific mRNAs deemed to promote disease growth, effectively silencing those segments of genes and preventing the usage of these mRNAs for protein synthesis, thus stimulating disease inhibition [26].

6.2.3 Factors Affecting Drug Release Rates The release of drug molecules from CaP NPs is dependent on several elements that can be manipulated to achieve maximum efficiency. Rates of drug release are mainly affected by pH environments, crystallinity, and the size and composition of the NPs. CaP remains dormant at basic pH levels, and a decrease in pH creates acidic environments, enhancing drug release rates [27]. Son and Kim [27] explained this conclusion in their study, loading anticancer drugs into CaP NPs released in varying pH environments, to which they found that the solution with the higher acidity (pH 4.5) had a faster release rate than a solution at a lower

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FIGURE 6–1 Schematic showing the mechanisms of intracellular uptake and release of CaP NPs. The CaP NPs (I) are illustrated to be taken into the cell by endocytosis (II), where their degradation in the endolysosome releases the encapsulated therapeutics (III, IV, V, VI) to their targeted destinations. CaP, Calcium phosphate; NP, nanoparticle. Reproduced from B. Neuhaus, B. Tosun, O. Rotan, A. Frede, A.M. Westendorf, M. Epple, Nanoparticles as transfection agents: a comprehensive study with ten different cell lines, RSC Adv. 6 (22) (2016) 18102 18112 [23].

acidity (pH 7.4). This is due to the CaP NP barrier dissolving faster in stronger and more acidic solutions, eliminating the extra step of drug particle diffusion through the CaP membrane. Thus the endolysosome is the ideal environment for CaP degradation, as its intracellular pH 5.5 is capable of effective CaP dissolution. Crystallinity is a factor referring to the extent of molecular organization in a material’s state of matter, and can be modified with reaction temperature alterations [28]. In a recent study, Zhang et al. [28] found that CaP NPs developed in solutions at 25 C had a higher drug release rate than CaP NPs developed at 80 C, indicating that increased crystallinity is proportional to decreased release rates. They also reported miniscule changes in the surface area and diameter of their CaP samples, suggesting that crystallinity adjustments only affected drug release rates, and not their structural properties.

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Particle size is another factor, as demonstrated in Dasgupta’s group [29]. When they increased their CaP NP diameters from 30 to 42 nm by increasing their reaction temperatures, they found that when loaded with bovine serum albumin, the larger NPs resulted in enhanced drug release rates, as explained by the rate of degradation being higher inside particles than on the surface; since larger particles also have an expanded interior, degradation occurs more rapidly and results in increased drug release [30]. The phase and composition of CaP NPs are arguably the most important influence on the rate of drug release. The ratio and purity of the two most widely accepted phases, hyaluronic acid (HA) and β-tricalcium phosphate (TCP), can be adjusted, where the dissolution and drug release rate of the NPs, by extension, increase in the presence of β-TCP [11]. Meanwhile, changing the composition could affect the Ca/P ratios of CaP. Two common forms of CaP used are hydroxyapatite (CaP ratio 1.67) and TCP (CaP ratio 1.50). Generally, a higher CaP ratio allows for better strength and resistance to degradation with minor exceptions, and can be fine-tuned to control the drug-delivery rate [31]. Composition alteration could also affect its charge density and surface charge. Hanifi et al. [32] added magnesium ions (Mg21) to their HA-β-TCP phase, which enhanced release rates due to the effects of β-TCP and the manipulation of particle size and crystallinity as a result of the increase in positive surface charges, when compared to control studies. In short, the ideal NP with the most efficient drug release rate would have a lower molecular weight and particle size, and a higher crystallinity.

6.3 Types of Calcium Phosphate Nanoparticles The flexibility of CaP NPs can be applied to synthesize diverse forms and shapes of NPs to adapt to different applications. To date, there are three main types of CaP NPs, each with noncoated and coated structures. Each structure has its own distinct advantages and disadvantages and fit for encapsulating different drugs. CaP NP structures are first introduced, followed by NP coatings. The merits and limitations of the different CaP NP structures are also discussed.

6.3.1 Bare Calcium Phosphate Nanoparticles 6.3.1.1 Core Core NPs are solid CaPs at nanoscale with therapeutic molecules conjugated to the surface or encapsulated in the core (Fig. 6 2A) [34]. It is synthesized by rapid mixing of two separate aqueous solutions with calcium and phosphate ions, then functionalized with therapeutics. They are specialized for protein and siRNA delivery. For example, Bakan et al. [33] synthesized and characterized spherical, pin-shaped, and calcium-deficient hydroxyapatite NPs for siRNA delivery using the sol gel technique. To increase siRNA loading, the NPs were functionalized with arginine (to increase transfection efficiency and reduce cytotoxicity) into the Ca21 precursor. Lyophilized siRNAs were then added into the CaP dispersion and incubated on a rotor to bind siRNA to the NPs. The exhibition of high therapeutic loading is the

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FIGURE 6–2 Bare CaP NPs and their existing structure variants. (A) The addition of arginine into the Ca21 component allowed for the improvement of siRNA loading of the HA. (B) The core shell structure of CaP NPs is encapsulated by a single solid shell and allows for a higher drug-loading capacity into the core. (C) Schematics of the core, core shell, and multilayer systems possible for CaP NPs for increasing biocompatibility and drug-loading efficiency capable of targeted intracellular delivery. CaP, Calcium phosphate; NP, nanoparticle. (A) From F. Bakan, G. Kara, M.C. Cakmak, M. Cokol, E.B. Denkbas, Synthesis and characterization of amino acid-functionalized calcium phosphate nanoparticles for siRNA delivery, Colloids Surf., B: Biointerfaces 158 (2017) 175 181 with permission [33]. (B) Reproduced from W. Jun, L. Lin, C. Yurong, Y. Juming, Recent advances of calcium phosphate nanoparticles for controlled drug delivery, Mini Rev. Med. Chem. 13 (10) (2013) 1501 1507.

prerequisite to developing nonviral vectors, which was proven valid by all the resultant NPs, with siRNA binding efficiency at as high as 90.0%. In vitro cytotoxicity tests on A549 cells also showed exceptional biocompatibility of CaP NPs, in which cell viability maintained over 90% after 72 h of incubation. This encourages further study of core-structured CaP NPs as therapeutic carriers. However, therapeutics attached to the surface are easily degradable by plasma enzymes, causing the NPs to exhibit low stability.

6.3.1.2 Core Shell Though core CaP NPs are effective at certain therapeutic deliveries, they still have several drawbacks, such as their relatively small surface area and capacity, that limit their further applications [2]. Core shell NPs are particles of drug molecules (core) encapsulated within a single layer of a solid shell (Fig. 6 2B) [35]. These are synthesized by mixing aqueous solutions composed of calcium ions, phosphate ions, and therapeutic molecules. The NPs can be loaded with a diverse range of therapeutics including biomacromolecules like siRNA

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and small hydrophilic drugs. For instance, Chen et al. [36] synthesized CaP NPs for siRNA delivery aiming at angiogenesis inhibition in breast cancer, where calcium chloride (CaCl2) and siRNA solutions were mixed and stirred with sodium citrate, resulting in NPs at 30.6 6 2.5 nm with clear hollow shells and siRNA encapsulated in the middle. No unbound siRNA was detected at a mass ratio of calcium chloride and siRNA of 15:1, indicating that the efficient binding between CaP and siRNA was reached. Resultant CaP NPs showed good gene-silencing capabilities, as evidenced by vascular endothelial growth factor (VEGF) mRNA expression as low as 19% when introduced to MCF-7 cell cultures. In vivo tests of NPs on mice models also demonstrated significantly smaller tumor sizes compared to the control group, suggesting this system to be a promising approach for breast cancer treatment. Core shell NPs can also be synthesized by reverse emulsion, as shown by Li et al. [37] with Triton X-100 and butanol as the emulsifier, and hexane as the oil phase. CaCl2 and siRNA with NaHPO4 solutions were dispersed and mixed to form core shell NPs for systematic siRNA delivery. The fabricated NPs showed no siRNA leakage at neutral pH, but released siRNA in 3 h under a weak acidic environment. In vitro and in vivo tests on the genesilencing effect of the synthesized NPs were able to halve luciferase activity in both H-460 cells and mouse specimens, showing the core shell NPs improved gene-silencing effects of siRNAs. These results encourage its potential application for clinical trials, but the significant shortcomings of core shell NPs include its lack of controlled drug release, as they are released quickly when the single shell degrades inside the endosome.

6.3.1.3 Multilayer Multilayer CaP NPs are the structure of multiple layers of CaP shells encasing a core, exhibiting higher stability and loading capacity compared to core shell or core structures in expense for larger sizes. Therapeutics, most commonly DNAs and RNAs, can be encapsulated within the core or spaced between shells, and released layer-by-layer over extended periods of time up to 24 h (Fig. 6 2C) [38]. In one study, Zhang et al. [39] synthesized siRNA-loaded multishell CaP NPs for gene silencing to circumvent the toxicity of viral vectors. CaNO3 and (NH4)2HPO4 were first rapidly mixed at pH 9 to form CaP cores, which were then dispersed with shRNA in water. The same process was repeated on the newly formed NPs three times, resulting in shRNA CaP multilayered NPs of 100 250 nm. Real-time polymerase chain reaction (PCR) results showed that the incorporation of the resultant CaP NPs to cells induced inhibition of osteocalcin expression. These results have suggested that multilayered CaP NPs can be used in tissue engineering for bone formation. In another study, Tobin et al. [40] synthesized triple-layered CaP NPs incorporating ultralow doxorubicin levels and siRNA for cancer treatment using similar procedures. Multilayered CaP NPs were first fabricated by precipitation of CaCl2 and Na2HPO4, before two more shells were added using the same reagents, resulting in triple-shelled CaP NPs of over 100 nm. The NPs were incubated with cancer cell lines with noncytotoxic levels of doxorubicin, paclitaxel, or etoposide for 2 h. In vitro cell uptake tests of the cancer cell lines showed as high as 64.5-fold amplification in the internalization of doxorubicin-loaded CaP NPs compared to the other drug groups, suggesting that the CaP NPs promoted caveolin-mediated endocytosis of cancer

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cells. These multilayered CaP NPs also produced greater gene silencing in vivo relative to siRNA concentration. Immunoblot densitometry of murine models injected with siRNAloaded CaP NPs were observed with an 80% reduction in the cross-linked inhibitor protein of apoptosis, and significant treatment effects could be observed at only 0.017 mg/m2 , compared to 30 mg/m2 in other studies. These results, combined with preferential localization of CaP NPs to tumors in vivo, encourages further study of modulating cellular endocytosic uptake for cancer therapy. Overall, core, core shell, and multilayered CaP NPs have distinct structures and are applied in different biomedical practices which most suitably utilize their advantages.

6.3.2 Coated Calcium Phosphate Nanoparticles It is well known that the pH value of endosomes of tumor cells are lower than that of normal cells. Naked CaP NPs as DDSs always release cargo at the pH of normal cells. Protective coatings can significantly increase the efficacy of CaP NPs, and, more importantly, decrease the threshold pH of CaP NP degradation, which provides the possibility of targeted drug delivery. At present, there are two main strategies. The first is lipid coating, which has a similar structure to plasma membranes to increase the cellular uptake of the CaP NPs. The other is polymer coating, which can form narrowly distributed hybrid NPs based on the selfassembly of CaP NPs with polymers to minimize the size of CaP NPs. For that reason, new delivery systems such as lipid- and polymer-coated CaP NPs have been developed for improved intracellular uptake and in vivo stability.

6.3.2.1 Lipid-Coated Calcium Phosphate Nanoparticles Lipid-coated CaP NPs (LCP NPs) have superior properties as a therapeutic carrier due to their plasma membrane-like composition, liposome-like structure, and modificative surface. Moreover, the cellular uptake can be improved significantly by means of the addition of positively charged lipid coatings. The LCP NPs are also popular due to their simple synthesis procedures. The CaP core can be prepared by microemulsion technology, followed by a coating of a lipid layer. As development methods of LCP NPs have improved over recent years, with the most current being codelivery systems, so too has drug-delivery efficiency seen drastic enhancements (Fig. 6 3). Huang’s group demonstrates the simplest development with CaP NPs coated with the lipid 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) [37]. These NPs were used on NCI-H-460 cells (human lung cancer cells) for the silencing of luciferase, and the results showed that luciferase expression was downregulated more than 60%, much higher than that of liposome polycation DNA at 20%. This group further coated the CaP NPs with an amphiphilic anionic lipid dioleoylphosphatidic acid (DOPA) [41]. It was found that the DOPA-coated CaP NPs showed a 10-fold improved silencing activity in vitro compared to the DOTAP-coated CaP NPs with a size of 150 nm [41]. This is attributed to the ion-pair formation between the negatively charged DOPA and the positively charged CaP NPs, limiting their sizes to within 25 30 nm. In another study, an optimized LCP NP delivery system was prepared by Tang’s group with

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FIGURE 6–3 Lipid-coated CaP NPs. Schematic of the recent development of lipid-coated CaP NPs. The drug-delivery efficacy increases from I to IV, and V is a newly developed codelivery system. (I) DOTAP-coated CaP NPs for siRNA delivery. (II) Negatively charged DOPA-coated (inner layer) and DOTAP-coated (outer layer) CaP NPs with 10-fold improved efficacy. (III) Antibody conjugated lipid-coated CaP NPs for targeted drug delivery. (IV) PEGylated lipidcoated CaP NPs with highly extended circulation time. (V) Codelivery system for phased release of various kinds of drugs. CaP, Calcium phosphate; DOPA, dioleoylphosphatidic acid; NP, nanoparticle.

DOPA and dioleoylphosphatidylcholine at a Ca/P ratio of 1.0, and the average particle size of 40 nm [42]. These CaP NPs were found to be more efficient delivering the functional CD siRNAs to MDA-MB-468 cancer cells, and inhibited cell growth in comparison to the commercial transfection reagent Oligofectamine (LifeTechnologies, Carlsbad, California, United States). It was also reported that the loading capacity of siRNA and its protection from enzyme degradation were significantly influenced by the Ca/P ratio. The loading capacity of LCP NPs increased with an increasing Ca/P ratio because of fewer phosphates at higher Ca/P ratios, and as such more DNA molecules were encapsulated by the CaP NPs. In contrast, nucleic acids loaded in NPs with a higher Ca/P ratio degraded more quickly, as the protection of the CaP core is based on the affinity between Ca21 ions and PO32 4 of the gene. In further works, they designed and developed an epidermal growth factor receptor-specific single-chain fragment antibody ABX-EGF scFv conjugated LCP NPs, and further enhanced the delivery efficacy of siRNA [43]. Another group of researchers, Chen and Watson, used solid core CaP-lipid NPs to encapsulate antisense oligonucleotides directed to the SOD1 gene, and used zebrafish as a model to monitor and study the distribution of the NPs in the brain, spinal cord, and blood circulation [44]. It was found that the hydrophilic PEGylated lipid coating was able to extend the circulation time of CaP NPs for up to 29 h, and the CaP NPs could protect the cargo more efficiently after diffusing through the blood brain barrier.

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In a recent study, LCPs have been combined with calcium carbonate (CaC) to fabricate LCP NPs for precisely controlled release at the endosomal pH of 5.5 6.0. These new NPs possess both high sensitivity and quick release under relatively mild acidic pH conditions, and, more importantly, were able to avert the loss of cargo in the blood during circulation in the tumor extracellular environment at pH 6.5 6.9 [45]. It is envisioned that the LCP NPs hold great potential as a gene-delivery vehicle with precise release properties at specific pH. From the procedure of synthesizing LCP NPs, researchers noticed the potential of LCP NPs for a codelivery system of multiple drugs for complementary and synergistic therapeutic efficacy. The codelivery system is essentially constructed in two steps: the encapsulation of therapeutic A in the LCP NPs, and the coencapsulation of therapeutic B in the lipid layer. For instance, Zhou et al. constructed a coencapsulation system to deliver and release multiple drugs sequentially, including hydrophilic miRi-221/222 (inhibitors for microRNA-221 and microRNA-222) inside CaP NPs and hydrophobic paclitaxel between CaP cores and lipid layers. It was found that this codelivery system had superior tumor cell suppression capabilities than the single DDS of paclitaxel [46]. These results have suggested that the LCP NP-based multidrug codelivery systems possess superior capabilities for multiple drug combinations with different water solubilities (hydrophilic and hydrophobic) and action mechanisms (gene therapy and chemotherapy). In another study, Chen et al. produced a stable core membrane siRNA delivery system with polycation liposomeencapsulated CaP NPs (PLCP) using 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [36]. This system was used to investigate the VEGF siRNA delivery efficiency of PLCP both in vitro and in MCF-7 xenograft mice. The study showed that VEGF siRNA mediated by PLCP represented superior capacity of gene silencing, resulting in significant angiogenesis and tumor growth inhibition [36].

6.3.2.2 Polymer Coating Although LCP NPs can promote systemic delivery of siRNA and DNA to tumor cells, potential toxicity of the organic solvents and the surfactants employed to prepare the CaP NPs raise significant concerns. To improve the biocompatibility of functionalized DDSs, polymercoated CaP NPs were therefore developed. To date, polyethylene glycol (PEG) and polyethylenimine (PEI) are the two most widely used coating polymers of CaP NPs. PEG functionalization increases colloidal stability and blood circulation time of CaP NPs by steric effects of the polymer layer, allowing for better control of targeted delivery [47]. In addition, PEG coatings formed from two dominant methods have superiority over lipid coatings with precise control over CaP NP size, which is critical for intracellular uptake, as well as its excellent biocompatibility (Fig. 6 4A). Pittella et al. [49] synthesized CaP NPs functionalized with block copolymers of PEG and charge-conversional polymers for gene knockdown. Polymer chains were grafted to CaP NPs by mixing the PEG-polyanion block copolymers, resulting in NPs of around 50 nm in diameter. Cell viability tests using PanC-1 cells showed near 100% viability at up to 1 μM siRNA, indicating that the hybrid NPs have excellent biocompatibility. Real-time PCR analysis of cancer cells incubated with free siRNA and CaP NPs for 3 h were also performed, and the functionalized CaP NPs showed 82% gene

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FIGURE 6–4 Polymer-coated CaP NPs. (A) Schematic of the formation of polymer-coated CaP NPs. Method 1 outlines the formation of irregularly shaped elongated PEGylated aggregated spheres from loosely aggregated networks; method 2 details the early presence of PEGylated chelators forming larger PEGylated needle bundles. (B) Schematic of the comparison of PEG- and PEI-coated CaP NPs. PEG coatings contribute to intracellular uptake and the alteration of NP size, while PEI coatings have more interaction between positively charged NPs and negatively charged cell membranes. CaP, Calcium phosphate; NP, nanoparticle; PEI, polyethylenimine. Reproduced from X. Huang, D. Andina, J. Ge, A. Labarre, J.-C. Leroux, B. Castagner, Characterization of calcium phosphate nanoparticles based on a PEGylated chelator for gene delivery, ACS Appl. Mater. Interfaces 9 (12) (2017) 10435 10445 [48].

knockdown while free-flowing siRNAs exhibited nearly 0% knockdown efficiency. This suggested that the PEG-coated CaP NPs are eligible to be alternative transfection agents to LCPs for siRNA delivery. In another study, Giger et al. synthesized core-shelled pDNA-CaP NPs functionalized with PEG-bisphosphonic acid [50]. Transfection experiments using HeLa cells showed that the PEG-functionalized NPs exhibited 65% transfection efficiency compared to the bare CaP NPs (40%), suggesting that the PEG coating promoted intracellular uptake of CaP NPs. Flow cytometry for 48 h was also performed to investigate the stability of NPs in cells. While bare NPs had negligible transfection efficiency after 48 h, these PEGcoated CaP NPs showed promising transfection efficiency of 65%, indicating that the PEG coating contributed to enhanced transfection of the CaP NPs. Finally, cell viability on HeLa

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cells was tested, and the PEG-coated CaP NPs exhibited an overall lower cytotoxicity than the bare NPs. These findings confirmed that PEG-coated CaP NPs are promising carriers for therapeutics. In contrast, PEI coatings add positive charges to the surface of CaP NPs so that they have a higher affinity to the negatively charged cell membranes and become preferably taken up by cells (Fig. 6 4B). In one study, Sokolova et al. [51] synthesized PEI-functionalized multilayered CaP NPs to enhance DNA transfection. The addition of PEI to the surface of NPs changed the zeta potential from 32 to 136 mV, promoting transfection efficiency of the CaP NPs in HeLa cells. Nearly a twofold increase was exhibited by the PEI-coated CaP NPs compared to the naked CaP NPs, and the luciferase activity multiplied by four compared to DNA/PEI, suggesting that the PEI coating was essential in maximizing transfection efficiency. In another study, Chernousova and Epple [52] compared the efficiency of PEI-coated CaP NPs and a commercial transfection reagent lipofectamine for siRNA delivery. In vitro transfection of HeLa cells showed that the expression of enhanced green fluorescent proteins loaded in the PEI-coated CaP NPs started after 5 h, compared to lipofectamine’s 4 h. The PEI-coated CaP NPs also exhibited a lower gene-silencing efficiency at 35%, compared to lipofectamine’s 100%. Despite the slower transfection time and lower gene-silencing efficiency, the cytotoxicity of PEI-coated CaP NPs allowed for better viability and proliferation of cells. The number of cells treated with PEI-coated CaP NPs increased by 700%, while lipofectamine only increased by 400% after 3 days of culture. These results show that while PEI-CaP NPs may not be as efficient as lipid-based transfection agents, their vastly superior biocompatibility allows for better in vivo applications while maintaining significant therapeutic effects, and therefore are comparable in competitiveness with LCP NPs. Despite the superior characteristics of PEG- and PEI-coated CaP NPs, there are also candidates for coating CaP NPs used for specific biomedical applications. In bone cancer metastasis treatment, uptake of therapeutics often occurs at nontargeted sites due to unique bone microstructures, thus the specialized functionalization of CaP NPs is needed for bone targeting. For example, Chu et al. [53] synthesized alendronate (ALN)-functionalized CaP NPs for the treatment of bone cancer metastasis. CaP NPs were first loaded with the drug methotrexate (MTX) and coated with ALN PEG, which has an excellent affinity to the exposed HA around metastasis sites. Isothermal titration calorimetry using ALN and PEG as positive and negative controls was performed, and the resultant CaP NPs showed a nearly identical affinity curve as ALN with bone tissues, suggesting they have bone-targeting capabilities similar to free ALN. In vitro anticancer assays showed that MTX-NPs have similar proliferation-inhibiting capabilities to MTX, both dropping MCF-7 cell viability to half at 1 mg/mL. The results showed that the efficacy of MTX-loaded NPs is slightly lower than that of MTX in small doses due to the time consumption for cellular uptake and drug release. Nevertheless, the enhanced bone-targeting and anticancer properties of the NPs suggest that ALN/PEG-MTX-CaP NPs are a promising platform for drug delivery in treating bone cancer metastasis. On the other hand, receptors of HAs such as CD44 were found overexpressed in certain cancer cells, and were widely utilized in targeted drug delivery [54]. For instance, Zhou et al. [55] synthesized HA/CaP NPs with a cationic CaP core and an anionic HA-SS-HA

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shell for siRNA delivery for melanoma cancer treatment. Fluorescence intensity of FAMsiRNA in cells decreases from 100% to 20% with increasing HA concentration used for interference, suggesting that the intracellular uptake of NPs was regulated by pathways affected by HA receptors. In vitro gene-silencing trials and western blots using B16F10 cells showed that the siLuc- and siBcl2-loaded HA-NPs have extremely high gene-silencing capabilities against luciferase and Bcl2, peaking at 85% and 83%, respectively. This DDS was also highly effective in vivo, lowering the luciferase activity in mice to 42% compared with the CaP NPs loaded with nonspecific control RNA, and reducing tumor weight to one-fourth that of nontreated mice. These results show that functionalization of HA was effective at promoting the efficacy of loaded therapeutics in CaP NPs.

6.4 Conclusion and Perspectives Thanks to the outstanding merits of biocompatibility, biodegradability, adsorption ability, adjustable structure, and ease of modification, CaP NPs have been regarded as one of the most promising DDSs [11]. Although the past several years have witnessed tremendous development and innovation in CaP NP-based DDSs, we must realize that there are still many scientific and technological challenges to be addressed. For example, morphological characteristics like pore sizes and pore-size distribution should be optimized since they play a crucial role in drug-loading quantity and its release kinetics [2,56]. Moreover, several factors related to the impregnation process including drug concentration, pH, duration, and pressure need to be taken into account because of their important effects on the amount of adsorbed drugs. Furthermore, the insufficient colloidal stability and rigid degradation in the physiological environment fail to meet the requirements for prolonged drug release, leading to unsatisfied therapeutic efficiency [9]. Therefore researchers have to put forward suitable approaches to shield cargo by coating lipids or polymers onto the CaP NPs. In addition, targeted drug delivery, efficient therapeutic dosage, and controlled drug release (release rates, release period, and continuous or on off release) on-site from CaP NP-based DDSs are still the main challenges that need to be understood and conquered for further biomedical applications [2]. For example, we should not only consider the adjustment of CaP NP drug interactions, but also the physicochemical characteristics of different CaP delivery systems and the drugs’ bioactivity at molecular or cellular level. In some cases, the functionalization of CaP NPs with specific molecules may be helpful to reach the targeted site, and the increase in the surface area is necessary to enhance the encapsulation of drugs [57]. More research endeavor should also be encouraged to fabricate multiresponsive controlled CaP NP-based DDSs. These triggers could either be in a separated or collaborative fashion, which result in an improved therapeutic effect of delivered drugs. For example, the combination of internal (pH) and external (light) contributions [58] was found to be able to achieve enhanced treatment effects with higher precision and accuracy. Specifically, the main challenges associated with CaP NPs for gene therapy such as their inefficient loading capabilities, poor intracellular entrance, physical stability, inadequate

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in vivo pharmacokinetics, and low nuclear transfection efficiency limit their therapeutic application [11]. Further efforts focused on exploring new methods or incorporating CaP NPs into other materials to stabilize the system and improve transfection efficiency have gained more attention and rapid development. For example, the development of multishelled, lipidor polymer-coated CaP NPs is a potential approach to address this issue [59]. In the near future, it is therefore important to establish a versatile carrier that could encapsulate different drugs to achieve an extended therapeutic window, thus realizing simultaneous multiphasedrug release and sequential-drug release from the carriers. Finally, researchers should be devoted to the construction of “theranostics” by integrating both imaging and therapy modalities with CaP NPs [60,61]. For example, CaP can be used to coat magnetic iron oxide (Fe3O4) to construct core shell NPs as efficient anticancer drug nanocarriers. Together with magnetic resonance (MR) imaging, the developed NPs were regarded as promising multifunctional nanodevices for nanotherapeutic approaches [62,63]. In addition, the CaP core is able to be a carrier for near-infrared fluorescent dyes and photosensitizers, which can be utilized as imaging tools, enabling localization in the body during photodynamic therapy of tumors, and enabling specialized peptides for targeted delivery to endothelial cells in tumors [64]. In such systems, these imaging techniques make the treatment process visible in real time, thus providing a more specialized therapy efficacy with reduced adverse effects [65]. To overcome these challenging tasks, interdisciplinary science involving materials, chemistry, biology, engineering, and medicine, as well as in-depth animal studies, should be set up, and more systematic knowledge on controlling nanoscale structures needs to be obtained. Over the coming years, it is expected that the next generation of CaP NP-based DDSs will be equipped with versatile and flexible qualities and that they will be able to function at the cellular level to combat various clinically identified devastating diseases.

Acknowledgments This work was supported by a start-up fund (1-ZE7S) and central research fund (G-YBWS) from the Hong Kong Polytechnic University. The authors would like to thank Elsevier publications for permission to reuse the referenced figures published by them.

References [1] K. Cho, X. Wang, S. Nie, D.M. Shin, Therapeutic nanoparticles for drug delivery in cancer, Clin. Cancer Res. 14 (5) (2008) 1310 1316. [2] W. Jun, L. Lin, C. Yurong, Y. Juming, Recent advances of calcium phosphate nanoparticles for controlled drug delivery, Mini Rev. Med. Chem. 13 (10) (2013) 1501 1507. [3] A. Kumari, S.K. Yadav, S.C. Yadav, Biodegradable polymeric nanoparticles based drug delivery systems, Colloids Surf., B: Biointerfaces 75 (1) (2010) 1 18. [4] J. Wen, K. Yang, F. Liu, H. Li, Y. Xu, S. Sun, Diverse gatekeepers for mesoporous silica nanoparticle based drug delivery systems, Chem. Soc. Rev. 46 (19) (2017) 6024 6045.

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[5] M.B. Zakaria, A.A. Belik, C.H. Liu, H.Y. Hsieh, Y.T. Liao, V. Malgras, et al., Prussian blue derived nanoporous iron oxides as anticancer drug carriers for magnetic-guided chemotherapy, Asian J. Chem. 10 (7) (2015) 1457 1462. [6] S. Han, A. Samanta, X. Xie, L. Huang, J. Peng, S.J. Park, et al., Gold and hairpin DNA functionalization of upconversion nanocrystals for imaging and in vivo drug delivery, Adv. Mater. 29 (18) (2017) 1700244. ´ [7] M. Jarosz, A. Pawlik, M. Szuwarzynski, M. Jaskuła, G.D. Sulka, Nanoporous anodic titanium dioxide layers as potential drug delivery systems: drug release kinetics and mechanism, Colloids Surf., B: Biointerfaces 143 (2016) 447 454. [8] D. Pastorino, C. Canal, M.-P. Ginebra, Drug delivery from injectable calcium phosphate foams by tailoring the macroporosity drug interaction, Acta Biomater. 12 (2015) 250 259. [9] A. Tabakovi´c, M. Kester, J.H. Adair, Calcium phosphate-based composite nanoparticles in bioimaging and therapeutic delivery applications, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 4 (1) (2012) 96 112. [10] M. Parent, H. Baradari, E. Champion, C. Damia, M. Viana-Trecant, Design of calcium phosphate ceramics for drug delivery applications in bone diseases: a review of the parameters affecting the loading and release of the therapeutic substance, J. Controlled Release 252 (2017) 1 17. [11] S. Bose, S. Tarafder, Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: a review, Acta Biomater. 8 (4) (2012) 1401 1421. [12] X. Xu, Z. Li, X. Zhao, L. Keen, X. Kong, Calcium phosphate nanoparticles-based systems for siRNA delivery, Regen. Biomater. 3 (3) (2016) 187 195. [13] T. Sun, Y.S. Zhang, B. Pang, D.C. Hyun, M. Yang, Y. Xia, Engineered nanoparticles for drug delivery in cancer therapy, Angew. Chem. Int. Ed. 53 (46) (2014) 12320 12364. [14] J.H. Woo, Y. Shimoni, W.S. Yang, P. Subramaniam, A. Iyer, P. Nicoletti, et al., Elucidating compound mechanism of action by network perturbation analysis, Cell 162 (2) (2015) 441 451. [15] L. Ansari, F. Shiehzadeh, Z. Taherzadeh, S. Nikoofal-Sahlabadi, A. Momtazi-Borojeni, A. Sahebkar, et al., The most prevalent side effects of pegylated liposomal doxorubicin monotherapy in women with metastatic breast cancer: a systematic review of clinical trials, Cancer Gene Ther. 24 (5) (2017) 189. [16] S. Kobayashi, M. Ueno, H. Hara, K. Irie, Y. Goda, S. Moriya, et al., Unexpected side effects of a high S-1 dose: subanalysis of a phase III trial comparing gemcitabine, S-1 and combinatorial treatments for advanced pancreatic cancer, Oncology 91 (3) (2016) 117 126. [17] J.-L. Huang, H. Chen, X.-L. Gao, Lipid-coated calcium phosphate nanoparticle and beyond: a versatile platform for drug delivery, J. Drug Target 26 (2017) 398 406. [18] C. Wu, J. Xu, Y. Hao, Y. Zhao, Y. Qiu, J. Jiang, et al., Application of a lipid-coated hollow calcium phosphate nanoparticle in synergistic co-delivery of doxorubicin and paclitaxel for the treatment of human lung cancer A549 cells, Int. J. Nanomed. 12 (2017) 7979. [19] C.S. Cummings, A.S. Campbell, S.L. Baker, S. Carmali, H. Murata, A.J. Russell, Design of stomach acidstable and mucin-binding enzyme polymer conjugates, Biomacromolecules 18 (2) (2017) 576 586. [20] A. Verma, S. Sharma, P.K. Gupta, A. Singh, B.V. Teja, P. Dwivedi, et al., Vitamin B12 functionalized layer by layer calcium phosphate nanoparticles: a mucoadhesive and pH responsive carrier for improved oral delivery of insulin, Acta Biomater. 31 (2016) 288 300. [21] L. Naldini, Gene therapy returns to centre stage, Nature 526 (7573) (2015) 351. [22] M. Epple, K. Ganesan, R. Heumann, J. Klesing, A. Kovtun, S. Neumann, et al., Application of calcium phosphate nanoparticles in biomedicine, J. Mater. Chem. 20 (1) (2010) 18 23. [23] B. Neuhaus, B. Tosun, O. Rotan, A. Frede, A.M. Westendorf, M. Epple, Nanoparticles as transfection agents: a comprehensive study with ten different cell lines, RSC Adv. 6 (22) (2016) 18102 18112.

162

THERANOSTIC BIONANOMATERIALS

[24] V. Sokolova, D. Kozlova, T. Knuschke, J. Buer, A.M. Westendorf, M. Epple, Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells, Acta Biomater. 9 (7) (2013) 7527 7535. [25] S. Behzadi, V. Serpooshan, W. Tao, M.A. Hamaly, M.Y. Alkawareek, E.C. Dreaden, et al., Cellular uptake of nanoparticles: journey inside the cell, Chem. Soc. Rev. 46 (14) (2017) 4218 4244. [26] D. Holoch, D. Moazed, RNA-mediated epigenetic regulation of gene expression, Nat. Rev. Genet. 16 (2) (2015) 71. [27] K.D. Son, Y.-J. Kim, Anticancer activity of drug-loaded calcium phosphate nanocomposites against human osteosarcoma, Biomater. Res. 21 (2017) 13. Available from: https://doi.org/10.1186/s40824-0170099-1. [28] H. Zhang, D. Yan, S.M.K. Gedara, S.S.F.D. Marakkalage, J.G.K. Methlal, Y. Han, et al., Effects of crystallinity and surface modification of calcium phosphate nanoparticles on the loading and release of tetracycline hydro-chloride, IOP Conf. Ser. Mater. Sci. Eng. 182 (1) (2017) 012052. [29] S. Dasgupta, A. Bandyopadhyay, S. Bose, Reverse micelle-mediated synthesis of calcium phosphate nanocarriers for controlled release of bovine serum albumin, Acta Biomater. 5 (8) (2009) 3112 3121. Available from: https://doi.org/10.1016/j.actbio.2009.04.031. [30] F. Alexis, Factors affecting the degradation and drug-release mechanism of poly(lactic acid) and poly [(lactic acid)-co-(glycolic acid)], Polym. Int. 54 (1) (2005) 36 46. Available from: https://doi.org/ 10.1002/pi.1697. [31] L. Sun, L.C. Chow, S.A. Frukhtbeyn, J.E. Bonevich, Preparation and properties of nanoparticles of calcium phosphates with various Ca/P ratios, J. Res. Nat. Inst. Stand. Technol. 115 (4) (2010) 243. [32] A. Hanifi, M.H. Fathi, H.M. Sadeghi, J. Varshosaz, Mg21 substituted calcium phosphate nano particles synthesis for non viral gene delivery application, J. Mater. Sci. Mater. Med. 21 (8) (2010) 2393 2401. Available from: https://doi.org/10.1007/s10856-010-4088-3. [33] F. Bakan, G. Kara, M.C. Cakmak, M. Cokol, E.B. Denkbas, Synthesis and characterization of amino acidfunctionalized calcium phosphate nanoparticles for siRNA delivery, Colloids Surf., B: Biointerfaces 158 (2017) 175 181. [34] D. Kozlova, S. Chernousova, T. Knuschke, J. Buer, A.M. Westendorf, M. Epple, Cell targeting by antibody-functionalized calcium phosphate nanoparticles, J. Mater. Chem. 22 (2) (2012) 396 404. [35] M.B. Gawande, A. Goswami, T. Asefa, H. Guo, A.V. Biradar, D.-L. Peng, et al., Core shell nanoparticles: synthesis and applications in catalysis and electrocatalysis, Chem. Soc. Rev. 44 (21) (2015) 7540 7590. [36] J. Chen, X. Sun, R. Shao, Y. Xu, J. Gao, W. Liang, VEGF siRNA delivered by polycation liposomeencapsulated calcium phosphate nanoparticles for tumor angiogenesis inhibition in breast cancer, Int. J. Nanomed. 12 (2017) 6075. [37] J. Li, Y.-C. Chen, Y.-C. Tseng, S. Mozumdar, L. Huang, Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery, J. Controlled Release 142 (3) (2010) 416 421. Available from: https://doi.org/10.1016/j.jconrel.2009.11.008. [38] R. Torabi, H. Ghourchian, M. Amanlou, P. Pasalar, Aptamer-conjugated calcium phosphate nanoparticles for reducing diabetes risk via retinol binding protein 4 inhibition, Can. J. Diabetes 41 (3) (2017) 305 311. [39] X. Zhang, A. Kovtun, C. Mendoza-Palomares, M. Oulad-Abdelghani, F. Fioretti, S. Rinckenbach, et al., SiRNA-loaded multi-shell nanoparticles incorporated into a multilayered film as a reservoir for gene silencing, Biomaterials 31 (23) (2010) 6013 6018. [40] L.A. Tobin, Y. Xie, M. Tsokos, S.I. Chung, A.A. Merz, M.A. Arnold, et al., Pegylated siRNA-loaded calcium phosphate nanoparticle-driven amplification of cancer cell internalization in vivo, Biomaterials 34 (12) (2013) 2980 2990. [41] J. Li, Y. Yang, L. Huang, Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for siRNA delivery to the tumor, J. Controlled Release 158 (1) (2012) 108 114.

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[42] J. Tang, L. Li, C.B. Howard, S.M. Mahler, L. Huang, Z.P. Xu, Preparation of optimized lipid-coated calcium phosphate nanoparticles for enhanced in vitro gene delivery to breast cancer cells, J. Mater. Chem. B 3 (33) (2015) 6805 6812. [43] J. Tang, C.B. Howard, S.M. Mahler, K.T. Thurecht, L. Huang, Z.P. Xu, Enhanced delivery of siRNA to triple negative breast cancer in vitro and in vivo through functionalizing lipid-coated calcium phosphate nanoparticles with dual target ligands, Nanoscale 10 (2018) 4258 4266. [44] L. Chen, C. Watson, M. Morsch, N.J. Cole, R.S. Chung, D.N. Saunders, et al., Improving the delivery of SOD1 antisense oligonucleotides to motor neurons using calcium phosphate-lipid nanoparticles, Front. Neurosci. 11 (2017) 476. [45] Y. Wu, W. Gu, J. Tang, Z.P. Xu, Devising new lipid-coated calcium phosphate/carbonate hybrid nanoparticles for controlled release in endosomes for efficient gene delivery, J. Mater. Chem. B 5 (34) (2017) 7194 7203. [46] Z. Zhou, C. Kennell, J.-Y. Lee, Y.-K. Leung, P. Tarapore, Calcium phosphate-polymer hybrid nanoparticles for enhanced triple negative breast cancer treatment via co-delivery of paclitaxel and miR-221/222 inhibitors, Nanomed. Nanotechnol. Biol. Med. 13 (2) (2017) 403 410. [47] J. Spadavecchia, D. Movia, C. Moore, C.M. Maguire, H. Moustaoui, S. Casale, et al., Targeted polyethylene glycol gold nanoparticles for the treatment of pancreatic cancer: from synthesis to proof-of-concept in vitro studies, Int. J. Nanomed. 11 (2016) 791. [48] X. Huang, D. Andina, J. Ge, A. Labarre, J.-C. Leroux, B. Castagner, Characterization of calcium phosphate nanoparticles based on a PEGylated chelator for gene delivery, ACS Appl. Mater. Interfaces 9 (12) (2017) 10435 10445. [49] F. Pittella, M. Zhang, Y. Lee, H.J. Kim, T. Tockary, K. Osada, et al., Enhanced endosomal escape of siRNA-incorporating hybrid nanoparticles from calcium phosphate and PEG-block charge-conversional polymer for efficient gene knockdown with negligible cytotoxicity, Biomaterials 32 (11) (2011) 3106 3114. [50] E.V. Giger, J. Puigmartí-Luis, R. Schlatter, B. Castagner, P.S. Dittrich, J.-C. Leroux, Gene delivery with bisphosphonate-stabilized calcium phosphate nanoparticles, J. Controlled Release 150 (1) (2011) 87 93. [51] V. Sokolova, S. Neumann, A. Kovtun, S. Chernousova, R. Heumann, M. Epple, An outer shell of positively charged poly(ethyleneimine) strongly increases the transfection efficiency of calcium phosphate/ DNA nanoparticles, Asian J. Mater. Sci. 45 (18) (2010) 4952 4957. [52] S. Chernousova, M. Epple, Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent, Gene Ther. 24 (5) (2017) 282. [53] W. Chu, Y. Huang, C. Yang, Y. Liao, X. Zhang, M. Yan, et al., Calcium phosphate nanoparticles functionalized with alendronate-conjugated polyethylene glycol (PEG) for the treatment of bone metastasis, Int. J. Pharm. 516 (1 2) (2017) 352 363. [54] R.E. Eliaz, S. Nir, F.C. Szoka Jr, Interactions of hyaluronan-targeted liposomes with cultured cells: modeling of binding and endocytosis, Methods Enzymol. 387 (2004) 16. [55] Z. Zhou, H. Li, K. Wang, Q. Guo, C. Li, H. Jiang, et al., Bioreducible cross-linked hyaluronic acid/calcium phosphate hybrid nanoparticles for specific delivery of siRNA in melanoma tumor therapy, ACS Appl. Mater. Interfaces 9 (17) (2017) 14576 14589. [56] K. Lin, C. Wu, J. Chang, Advances in synthesis of calcium phosphate crystals with controlled size and shape, Acta Biomater. 10 (10) (2014) 4071 4102. [57] M.S. Lee, J.E. Lee, E. Byun, N.W. Kim, K. Lee, H. Lee, et al., Target-specific delivery of siRNA by stabilized calcium phosphate nanoparticles using dopa hyaluronic acid conjugate, J. Controlled Release 192 (2014) 122 130. [58] M. Gou, S. Li, L. Zhang, L. Li, C. Wang, Z. Su, Facile one-pot synthesis of carbon/calcium phosphate/ Fe3O4 composite nanoparticles for simultaneous imaging and pH/NIR-responsive drug delivery, Chem. Commun. 52 (74) (2016) 11068 11071.

164

THERANOSTIC BIONANOMATERIALS

[59] M.R. Mohammadi, A. Nojoomi, M. Mozafari, A. Dubnika, M. Inayathullah, J. Rajadas, Nanomaterials engineering for drug delivery: a hybridization approach, J. Mater. Chem. B 5 (22) (2017) 3995 4018. [60] Z. Yang, J. Ren, Z. Ye, W. Zhu, L. Xiao, L. Zhang, et al., Bio-inspired synthesis of PEGylated polypyrrole@polydopamine nanocomposites as theranostic agents for T 1-weighted MR imaging guided photothermal therapy, J. Mater. Chem. B 5 (5) (2017) 1108 1116. [61] W. Zhu, Y. Liu, Z. Yang, L. Zhang, L. Xiao, P. Liu, et al., Albumin/sulfonamide stabilized iron porphyrin metal organic framework nanocomposites: targeting tumor hypoxia by carbonic anhydrase IX inhibition and T1 T2 dual mode MRI guided photodynamic/photothermal therapy, J. Mater. Chem. B 6 (2) (2018) 265 276. [62] W.-M. Li, S.-Y. Chen, D.-M. Liu, In situ doxorubicin CaP shell formation on amphiphilic gelatin iron oxide core as a multifunctional drug delivery system with improved cytocompatibility, pH-responsive drug release and MR imaging, Acta Biomater. 9 (2) (2013) 5360 5368. [63] Y.-P. Wang, Y.-T. Liao, C.-H. Liu, J. Yu, H.R. Alamri, Z.A. Alothman, et al., Trifunctional Fe3O4/CaP/alginate core shell corona nanoparticles for magnetically guided, pH-responsive, and chemically targeted chemotherapy, ACS Biomater. Sci. Eng. 3 (10) (2017) 2366 2374. [64] K. Haedicke, D. Kozlova, S. Gräfe, U. Teichgräber, M. Epple, I. Hilger, Multifunctional calcium phosphate nanoparticles for combining near-infrared fluorescence imaging and photodynamic therapy, Acta Biomater. 14 (2015) 197 207. [65] L. Zhang, R. Liu, H. Peng, P. Li, Z. Xu, A.K. Whittaker, The evolution of gadolinium based contrast agents: from single-modality to multi-modality, Nanoscale 8 (20) (2016) 10491 10510.

7 Graphene and Graphene Oxide for Tissue Engineering and Regeneration Luoran Shang1, Yaping Qi1,2, Haiwei Lu1,3, Hao Pei1, Yiwei Li4, Liangliang Qu1, Zhengwei Wu1,5, Weixia Zhang1 1

JOHN A. PAUL SON S CHOOL OF ENGINEERING AND AP PLIED S CIENCE S, HARVARD UNIVERSITY, CA MBRIDGE, M A, UNITED STATES 2 DEPARTMENT OF PHYSICS, THE UNIVERSITY OF HONG KONG, POKFULAM, HONG K ONG 3 STATE KEY LABORATORY FOR MANUFACTURING SYSTEMS ENGINEERING, XI’AN JIAOTONG UNIVERSITY, X I’AN, P.R . C HINA 4 DEPART ME NT OF MECHANI CAL ENGINEERING, MASSAC HUSET TS INSTITUTE OF TECHNOLOGY, C AMBRIDGE, MA , UN I T ED STAT ES 5 DEPART ME NT OF BIOMEDI CAL ENGINEERING AND BIOTECHNOLOGY, UNIV ERSIT Y OF M AS SACHUSE TTS LO WELL , L OWELL , M A, UNIT ED STATE S

7.1 Introduction Graphene is a single layer of sp2 bonded carbon atoms arranged in a hexagonal lattice, which is a 2D building element of many other allotropes of carbon with different dimensionalities, such as 0D Bucky balls, 1D carbon nanotubes, and 3D graphite [1]. Since its first experimental discovery and characterization in 2004, graphene has become a rapidly rising star on the horizon of materials science [2]. Graphene and its major chemical derivative, graphene oxide (GO), have been intensively investigated over the last decade and a half. It has been demonstrated that graphene and GO have a large theoretical specific surface area, high Young’s modulus, excellent thermal and electrical conductivities, and unique optical properties [3]. These unusual properties quickly rendered graphene and GO for a tremendous number of applications in nanoelectronics, nanocomposites, sensors, supercapacitors, and energy storage in the early stage of graphene research [2,4 8]. Graphene-based materials were first introduced into the biomedical field in 2008, when Dai et al. conducted pioneer works by using GO as an efficient carrier for drug delivery [9,10]. Since then, intensive efforts have been devoted to exploring biomedical applications of graphene-based materials, ranging from drug/gene delivery [9,11,12], to bioimaging/ biosensing [13 15], to cell growth behavior control [16,17], and to biomedical devices for tissue engineering and regeneration [18,19]. Considering the rapid development in this challenging and dynamic field, it is necessary to overview the achievements in this field with

Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00007-9 © 2019 Elsevier Inc. All rights reserved.

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the most recent progresses. In this chapter, we focus specifically on the applications of graphene and GO in tissue engineering and regeneration, and summarize and critically appraise advancements in this direction. Although we do not cover other directions, detailed discussion can be found elsewhere in several excellent review articles [1,20 23]. Tissue engineering is a scientific field to reproduce or regenerate damaged tissues or whole organs [24]. To achieve its goal, it uses a combination of cells, engineering scaffolds, and biologically active molecules to assemble functional constructs that restore, maintain, or improve tissue function [25]. Therefore, developing novel suitable scaffolds to fabricate functional complex tissue constructs is crucial in tissue engineering. An ideal scaffold can carry active biomolecules, generate proper physiological signals, stimulate mechanical properties of the native tissue, and provide a substrate to interface with living cells, guiding cell attachment, proliferation, and differentiation [1,19]. Graphene and GO, as 2D materials with large surface areas, can provide sufficient substrates for cellular interaction and can carry many biomolecules, including DNA, enzymes, proteins, or peptides, through either covalent bond or noncovalent interactions, such as π π stacking. In addition, their exceptional mechanical and electrical properties enable these 2D materials to enhance the mechanical strength of tissue substitutes and to apply electrical signals. Hence, graphene and GO are excellent candidates for tissue engineering. In this chapter, we overview the applications and the recent progresses of graphene and GO in tissue engineering. To give better instruction into exploring potential applications of graphene-based materials for tissue engineering, this chapter is organized in a propertyoriented structure, which is different to other articles with an application-oriented structure. We mainly focus on mechanical, electrical, chemical, and other properties, such as morphology and impermeability of graphene and GO, and discuss how to apply these outstanding properties in tissue engineering fields, including bone, skin, muscle, cardiac, and neural tissue engineering, as shown in Fig. 7 1. In addition, since stem cells are widely used in tissue engineering, we also include stem cells in this chapter and discuss the impact of graphene and/or GO on proliferation and differentiation of stem cells for different applications in tissue engineering fields.

7.2 Properties and Applications in Tissue Engineering Graphene derives its outstanding properties from its unique chemical structure. In graphene, carbon atoms are sp2 hybridized. The sp2-hybridization is the combination of one s-orbital with only two p-orbitals to form three new sp2 hybrid orbitals that contribute together to have a planar configuration with a characteristic angle of 120 degrees. There is an additional p-orbital perpendicular to sp2 hybrid orbitals in each carbon atom. Each carbon atom uses these sp2 hybrid orbitals to form three covalent σ-bonds (C C bonds) with adjacent three carbon atoms. The planar configuration of sp2 hybrid orbitals determines the perfectly planar nature of graphene with a huge specific surface area of approximately 2630 m2/g [26], and the strong strength of C C bonds gifts graphene strong mechanical properties with a

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FIGURE 7–1 The overall tissue engineering applications of graphene and GO based on their properties. GO, Graphene oxide

Young’s modulus of 1100 GPa and a fracture strength of 130 GPa [27]. The additional perpendicular p-orbital forms a π-bond between two carbon atoms. The π-bonds on both sides of the graphene planar form a large delocalized conjugated system, enabling graphene to have exceptional thermal and electrical conductivities. It has been reported that graphene has a thermal conductivity of 5000 W/m/K [28], and an electrical conductivity of 10,000 S/cm [29], combining with an ultrahigh intrinsic mobility of 200,000 cm2/v/S [30,31]. In addition, the electronic structure of graphene also enables further chemical modification. The highly dense π electrons on the graphene plane can interact with many biomolecules containing aromatic structures through π π stacking [32]. They are also suitable for electrophilic reactions such as click reactions, cycloadditions, and carbine insertion reactions [33]. The hydrophobic graphene can also absorb various organic molecules or polymers with high hydrophobic character via van der Waals interaction [34]. For GO, besides the above noncovalent π π stacking and van der Waals interaction, abundant oxygen functional groups can be used to functionalize GO with various molecules or biomolecules through both noncovalent interactions including hydrogen bonds and ionic interactions, and covalent

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bonds with chemical reactions. Thus, graphene and GO can be easily combined with a variety of bioactive materials to obtain desired characteristics that can meet the requirements of tissue engineering. This section summarizes and discusses graphene and GO applications in the tissue engineering field based on their mechanical, electrical, chemical, and other properties.

7.2.1 Mechanical Properties and Applications The astonishing mechanical properties of graphene and GO can be used to prepare graphene-based composites with enhanced mechanical strength for tissue engineering applications. Bioceramics, hydrogels, films, fibers, and many other tissue engineering scaffolds can be greatly improved in mechanical properties and stability by using graphene and GO as reinforcement materials and, thus, can be applied in various tissue engineering fields. Graphene- or GO-enhanced biomaterials have been widely utilized in bone tissue engineering. With graphene or GO incorporated, these biocomposites have been greatly improved in mechanical strength. While it is well known that stiff substrates can promote bone differentiation [35], graphene-based composites can enhance osteogenic differentiation [35,36]. For example, hydroxyapatite (HA) is the major inorganic part of bone, which can support bone regeneration. After being incorporated with GO, HA/silk fibroin (HA/SF) composites had better mechanical properties with higher compressive strength and modulus, and GO HA/SF had been demonstrated to promote attachment and proliferation of mouse mesenchymal stem cells (MSCs) and to stimulate expression of the osteogenic gene osteocalcin, thus promoting differentiation of MSCs into bone [37]. GO has also been reported to significantly boost the tensile strength of poly(L-lactic-co-glycolic acid) (PLGA) and HA nanofibrous matrices. The PLGA/GO/HA matrices could serve as mechanically stable scaffolds for cell growth and could functionally promote alkaline phosphatase activity, the osteogenesisrelated gene expression, and mineral deposition, acting as excellent and versatile scaffolds for applications in bone tissue engineering (Fig. 7 2) [38]. Other similar research works about GO-enhanced HA composites have also been reported for bone tissue engineering [39 41]. Besides HA, graphene and GO have also been reported to enhance the mechanical properties of hydrogels and biopolymers. For instance, chemically converted graphene was mixed with cellulose to prepare self-supporting graphene hydrogel films that could stimulate osteogenic differentiation of stem cells, without additional inducer, both in vitro and in vivo [42]. In addition, a GO coating could improve the biomedical properties of collagen scaffold, including surface structure, compressive strength, and osteoblastic cell ingrowth, with enhanced biocompatibility and biodegradability [43]. In addition, the incorporation of GO significantly improved the compressive modulus of SF scaffolds, and GO/SF exhibited obviously enhanced osteogenic proliferation of osteoblastic cells compared to SF scaffolds without GO [44]. It has also been reported that a very low concentration of GO could reinforce biocompatible polymer polypropylene fumarate with a significantly enhanced compression and flexural strength, which was suitable for applications in bone tissue engineering [45]. What is more, various graphene-coated organic and inorganic substrates have shown

FIGURE 7–2 The PLGA/GO/HA matrices as scaffolds for cell proliferation and osteogenic differentiation. PLGA, PLGA/HA, and PLGA/GO matrices were used as controls. (A) Stress strain curves of different nanofibrous matrices. (B) Proliferation of MC3T3-E1 cells cultured on the different nanofibrous matrices for 1 7 days in vitro. (C) Fluorescent staining observation of MC3T3-E1 cells cultured on different nanofibrous matrices for 4 days: MC3T3-E1 cells have been cultured on nanofibrous matrices with cytoskeleton (FITC, green) and nucleus (DAPI, blue) staining. (D) Alizarin Red staining of MC3T3-E1 cells cultured on different nanofibrous matrices at 21 days. (E) ALP activities of MC3T3-E1 cells on different nanofibrous matrices during 14-day in vitro culture. (F) Calcium deposition after culturing on different nanofibrous matrices for 14 and 21 days; quantitative real-time PCR analysis of osteogenesis-related gene expression of RUNX2 (G) and OPN (H) after MC3T3-E1 cells cultured for 7 days. ALP, Alkaline phosphatase; PLGA, poly(L-lactic-co-glycolic acid); GO, graphene oxide; HA, hydroxyapatite. Reprinted with permission from C. Fu, H. Bai, J. Zhu, Z. Niu, Y. Wang, J. Li, et al. Enhanced cell proliferation and osteogenic differentiation in electrospun PLGA/hydroxyapatite nanofibre scaffolds incorporated with graphene oxide, PLoS One 12 (2017) e0188352.

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the capability of controlling and accelerating osteogenic differentiation of human MSCs, which was attributed to graphene’s exceptionally high Young’s modulus and its remarkable flexibility for out-of-plane deformation [46]. In addition to mechanical strength enhancement, graphene can also improve the toughness of hybrid materials for load-bearing implant applications to regenerate bone tissue. Natural bone, as a major load-bearing part, undergoes microcracking under everyday normal physiological load that stimulates the bone remodeling process to stop propagation of microcracks and finally makes new bones at the affected area. This toughening mechanism can be mimicked using graphene-based composites. The strong interaction between graphene or GO and matrix materials can bridge cracks and impede crack propagation, resulting in enhanced toughness [47]. For instance, it has been reported that GO functionalized poly (vinyl alcohol) (PVA) made the PVA not only stronger but also tougher. The tensile strength and Young’s modulus of the PVA films containing GO were increased significantly by 88% and 150%, respectively, and the elongation at break was also increased by 22%, as compared to those of the unfilled PVA [48]. Thus, the incorporation of graphene or GO can render brittle materials suitable for bone implant applications. Another major tissue engineering application related to mechanical enhancement of graphene-based materials is in the skin and muscle field. To stimulate skin and skeletal muscle regeneration, it is important to mimic the mechanical microenvironment of native extracellular matrix (ECM) using biomaterials. However, most existing biomaterials cannot mimic the mechanical behavior of ECM [49]. For example, hydrogels were widely employed as a class of ECM-mimicking materials as their hydrophilic polymeric network structure resembled ECM and their physicochemical properties could be well adjusted. However, it was very challenging to improve hydrogel’s toughness while maintaining its rigidity [50]. In addition, electrospun nanofibers possessed a nanoporous structure and thus high specific surface area, which was similar to ECM’s nanotopology. However, some of the materials show intrinsic brittleness, low toughness, as well as wear resistance, which largely limited their application as tissue engineering scaffolds [38]. Meanwhile, the addition of graphene or GO to biomaterials is able to tune the mechanical properties of biomaterials to match the mechanical behavior of ECM. Du et al. reported that reduced GO (rGO) addition could efficiently tailor the elastomeric behavior and mechanical strength of poly(citric acidoctanediol-polyethylene glycol) (PCE) composites. The enhancement of mechanical properties was attributed to the strong interactions between rGO and polymer matrix, and the mechanical properties and elastomeric behavior of PCE/GO nanocomposites were well matched with native skeletal muscle tissues, leading to significantly enhanced myogenic differentiation and regeneration [49]. Similarly, incorporation of rGO into polyacrylamide (rGO/ PAAm) hydrogels or PLGA/collagen hybrid matrices increased the Young’s moduli and elasticity of the hydrogels or matrices due to the strong molecular interactions between GO and polymer chains. The Young’s moduli of these hydrogels or matrices could be tuned by changing concentrations of graphene materials to mimic the mechanical properties of native skeletal muscle ECM, which was beneficial for enhanced differentiation of myoblasts into myotubes [51,52]. Patel et al. reported graphene-containing poly(ε-caprolactone) (PCL)

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nanocomposites to induce multinucleated myotube formation. They demonstrated that the mechanical properties of the nanocomposite scaffolds could be finely tuned by varying the graphene concentration, conferring cell-instructive ability to induce myoblast differentiation into multinucleated elongated myotubes even without differentiation media [53]. Moreover, the flexibility of graphene and GO combined with their unique electromechanical properties makes them suitable to fabricate artificial muscle [54 57]. Conventional rigid artificial devices have a mechanical mismatch with soft tissues, resulting in a low signal-to-noise ratio and/or mechanical fatigue and scarring [58]. Therefore, most conventional artificial muscles have a critical drawback of poor sustainability under long-term excitations, mainly due to the leakage of electrolytes and hydrated cations through cracks in the metallic electrodes [56]. Meanwhile, graphene can improve mechanical flexibility and stability under large bending deformation of the electrode without using any additional rigid metallic current collector. Graphene-based materials have also been used to improve the regeneration of poorly healing wounds. Nyambat et al. fabricated genipin-cross-linked adipose stem cell-derived ECM-nano GO composite sponge for skin tissue engineering. Typically, the mechanical properties of cell-derived ECM scaffolds are poorer than those of tissue-derived ECM scaffolds. By using GO and genipin-cross-linking to improve the poor mechanical properties of pure cell-derived ECM, they successfully developed a biomimetic ECM sponge and used it as a skin graft [59]. GO incorporated in collagen fibrin (CF) composite films were used as wound dressing materials for both in vitro and in vivo studies. The presence of GO increased the mechanical strength of CF composite films, thus enhancing wound healing [60]. Li et al. used three-dimensional graphene foams (3D-GF) loaded with bone marrow-derived MSCs to promote skin wound healing with reduced scarring. They demonstrated the bone marrowderived MSCs combined with 3D-GF biomechanical and biochemical features synergized to provide a better wound healing environment by enhancing early vascularization and reducing scarring [61]. Similar to artificial muscle applications, Hou et al. reported a strong and stretchable self-healing film containing graphene for potential artificial skin applications. The film exhibited greatly enhanced strain and tensile strength and a highly stable sensitivity to a very light touch, even in bending or stretching states. They demonstrated that this thin film could mimic both the mechanical self-healing and pressure sensitivity behavior of natural skin without any external power supply [62]. In addition, mechanically enhanced graphene-based materials have been used in other tissue engineering fields. In the cardiac field, myocardial infarction (MI) is one of the leading causes of morbidity and mortality worldwide, and numerous hydrogels designed with a broad range of mechanical properties have been employed to rebuild myocardial function, and most studies have shown that a stiffer hydrogel was more appropriate for rebuilding cardiac function [63]. For example, the addition of pristine graphene into collagen increased the stiffness to match that of the native cardiac tissue, resulting in enhancing human cardiac fibroblast growth, while simultaneously inhibiting bacterial adhesion on the biohybrids, since the graphene caused mechanical disruption of bacterial cells [64]. Introducing GO nanoparticles into oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels provided better mechanical support and gap junctions, leading to the enhanced generation of cytoskeletal structures as

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well as the formation of intercalated disk that is important for cardiac integrity and function [65]. Similarly, the presence of rGO could also significantly enhance the mechanical properties of gelatin methacryloyl (GelMA) hydrogels, and the rGO-GelMA scaffold acted as a more natural microenvironment for the cardiomyocytes and exhibited better biological activities such as cell viability, proliferation, and maturation [66]. What’s more, GO-incorporated methacryloyl substituted recombinant human tropoelastin (MeTro) has been reported to develop an elastic biomaterial for cardiac tissue engineering. The addition of GO improved both elastic modulus and rupture stain. The MeTro/GO gel exhibited remarkable resistance against rotation stress without any plastic deformation [67]. Interestingly, it has also been reported that introducing GO into multiarmed polyethylene glycol/melamine hydrogel via π π conjugation stacking resulted in a softer but mechanically stable hydrogel that is beneficial for transmitting mechanical signal and coordinating the beat of the myocardium tissue, thereby highly efficiently promoting the rebuilding of cardiac function in MI [63]. In addition, GO could also induce spontaneous cardiac differentiation in embryoid bodies (EBs) by changing the EBs’ stiffness [68]. In the neural tissue engineering area, although the most important advantage of graphene-based materials is their electrical properties, which we will discuss in the following section, the mechanical properties of these materials also enable their applications in neural tissue engineering. First, graphene-based materials can provide a suitable scaffold for neural cell culture. Graphene nanofibers prepared via electrospinning methods had a Young’s modulus similar to natural collagen fibers, providing an ideal mechanical match when acting as a soft matrix for the living cell culture and soft tissue regeneration, such as for brain and nerve [69]. Unexpectedly, the presence of rGO reduced significantly the mechanical stability of PCL/rGO membranes during the hydrolytic degradation process, but the PCL/rGO membrane still provided sufficient mechanical properties to theoretically comply with the specifications of neural tissue regeneration [70]. In addition, flexible graphene allowed to fabricate graphene-based devices for neural imaging and optogenetic applications. These devices were superior to the present indium tin oxide (ITO)-based electrode technology for its dramatically increased mechanical flexibility [71]. Therefore, due to the strong interaction between graphene materials and biomaterials as tissue engineering scaffolds, incorporation of graphene and GO can greatly enhance the mechanical strength of these scaffolds that could match the mechanical properties of native tissues, resulting in enhanced specific differentiation of stem cells for different tissue engineering applications. In addition, graphene-based materials can improve the toughness and flexibility of biohybrids so that these hybrids could be used to fabricate biomedical devices for either load-bearing implants in bone regeneration or stretchable devices in other tissue engineering fields.

7.2.2 Electrical Properties and Applications The conductive nature of graphene-based materials brings in good conductivity of many biohybrids that can be used for specific electrical signal-related tissue engineering applications.

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Due to the electrical characteristics of the neural system, the electrical properties of graphene-based biohybrids have been utilized in the neural tissue engineering field. Graphene as a scaffold has been reported to promote the adhesion and neural differentiation of human neural stem cells (hNSCs). A possible mechanism was attributed to the electrical coupling between graphene and the hNSCs, which could affect the bioelectricity of NSCs to promote the maturation of NSCs [72,73]. rGO also could be used to induce neural differentiation of MSCs, as reported by Guo et al. They fabricated a poly(3,4-ethylenedioxythiophene) (PEDOT) 2 rGO hybrid microfiber as a scaffold for a self-powered electrical stimulationassisted neural differentiation system for MSCs [74]. Li et al. utilized 3D graphene films as a robust scaffold for NSC culture in vitro. They proved that the 3D architecture of graphene films could effectively improve the electrical stimulation performance of the conductive scaffold for differentiated NSCs [75]. Besides neural differentiation, the high electrical conductivity of graphene could lead to promotion of neurite sprouting and better neurite outgrowth, as reported by several research groups [17,76,77]. And Liu et al. constructed a hydrogel by chemical crosslinking rGO and poly(ethylene glycol)-functionalized carbon nanotube (CNTPEG) with OPF polymer chains, and demonstrated that the rGO-modified hydrogel was electrically conductive and could enhance nerve cell responses [78]. What is more, the electrical properties of graphene and rGO enable to fabricate various biomedical devices for detecting neural signals or stimulating the neuronal activity [79]. Tang et al. investigated the impact of graphene on the formation of a functional neural network. They demonstrated graphene could support the growth of functional neural circuits and improve neural performance and electrical signaling in the neural networks [80]. Feng et al. reported on graphene nanofiber based on controllably assembled rGO, neurons expressed more branches of neurites and a faster growth rate. And they demonstrated that an unprecedented accelerated growth and development of neurons was achieved by using the graphene nanofibers for cellular electrical stimulation in a long-term culture period (Fig. 7 3) [69]. In addition, rGO was also used to fabricate an implantable neural electrode to detect the electrophysiological and neurochemical signaling in vivo. An rGO/Au2O3 nanocomposite-coated electrode exhibited a fast response to H2O2 with a very low detection limit, making it a rapid and reliable sensing platform for practical H2O2 detection in the brain or for other neural chemical molecules in vivo [81]. Cardiac tissues are electrically conductive, and the graphene-based composites own enhanced conductivity that can match the conductive properties of cardiac tissues or provide electrical stimulation for cardiac repair, thus they have many applications in the cardiac tissue engineering field. In vivo studies have shown that the electrical conductivity of graphene-based materials could upregulate the expression of cardiac specific markers, significantly improving the repair efficiency of several heart functions [63,65,82,83]. Conductive graphene-based scaffolds could further promote the cardiac differentiation and beating behavior of the EBs and increase the metabolic activity of cardiomyocytes to realize better cardiac functions, when applying electrical stimulation [64,66]. The excellent electrical properties of graphene-based materials have also been widely used in skin and muscle tissue engineering. The conductive nature of graphene-based

FIGURE 7–3 Soft graphene nanofibers (G-NFs) for the acceleration of nerve growth and development. (A, B) Fluorescent images of motor neurons after 3 days in culture on different substrates for (A) neuritogenesis [red: the neuronal marker protein of III β-tubulin (Tuj) for filopodia, green: dendrite marker protein of the microtubule-associated protein-2 (Map) for neurites]; and (B) cell maturation (red: neuraxon marker protein of tau expression). (C) Neurites elongation: maximum and mean lengths of neurites. (D) Neurites sprouting: the mean number of neurites and the branches of neurites. Blue: nuclear. (E) The relative fluorescence intensity change ΔF/F of the neuron during a pulse period, and the fluorescence images of the cells preincubated with Fluo-4AM (membrane-permeable and Ca21-dependent dye) on G-NFs before and after the pulse. Green: Ca21. Scale bars: (A), 80 μm; (B), 200 μm; and (E), 10 μm. Reprinted with permission from Z.Q. Feng, T. Wang, B. Zhao, J. Li, L. Jin, Soft graphene nanofibers designed for the acceleration of nerve growth and development, Adv. Mater. 27 (2015) 6462 6468.

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hybrids is allowed to provide electrical stimulation that can be combined with the improved mechanical strength to further enhance differentiation of myoblasts into myotubes. For example, incorporation of rGO into PAAm hydrogels not only enhanced mechanical properties of the hydrogel to match the mechanical behavior of native tissue, but also made the hydrogel electrically conductive so that additional electrical stimulation could be applied to myoblasts to significantly enhance the myogenic gene expression, making these graphenebased conductive and soft hydrogels ideal skeletal muscle tissue scaffolds that can simultaneously deliver electrical and mechanical cues to biological systems [51]. Similar results have been also reported with native graphene and rGO as substrates [84,85]. For artificial skin and artificial muscle applications, the electrical conductivity of biohybrid scaffolds is a very important factor, since most artificial skins or muscles require a thermoelectric or electromechanical response. Hou et al. prepared an rGO foam as an electronic skin for sensing human touch. The rGO foam was temperature-sensitive based on the thermoelectric effects of graphene. They demonstrated the pressure-sensing behaviors of the rGO foam under finger pressure according to finger heating effects, enabled human touch locating and pressure level measuring under zero working voltage [86]. Rogers et al. presented findings on a potential artificial muscle material based on monolayer GO that exhibited a fast response upon an electrical stimulus [54]. Most artificial muscles are electromechanically active actuators. Kotal et al. fabricated ionic polymer actuators as high-performance ionic artificial muscles using sulfur and nitrogen co-doped rGO. They demonstrated the excellent actuation performances of the artificial muscle were attributed to the ultrahigh capacitance of the sulfur and nitrogen co-doped rGO [57]. Zang et al. fabricated a novel artificial muscle actuator using a laminate of crumpled graphene and dielectric elastomer. When applying a direct-current voltage of 3000 V between the graphene films, the elastomer developed an electric field that induced the Maxwell stress. The stress deformed the laminate by reducing its thickness and increasing its area over 100%. They demonstrated the actuation was fast and the graphene elastomer laminate could restore its original state once the voltage was removed [55]. The conductive composites containing graphene or rGO can promote neural differentiation of hNSCs due to electrical coupling between graphene and hNSCs. The electrical conductivity of graphene-based composites can upregulate the expression of cardiac specific markers to improve the cardiac repair efficiency. Moreover, the conductive composites offer opportunities to provide electrical stimulation, which can induce respective differentiations for different tissue engineering applications and enhance metabolic activity of both nerve cells and cardiomyocytes. The conductive composites can also be used to fabricate biosensors and artificial skins and muscles.

7.2.3 Chemical Properties and Applications The chemical properties of graphene-based materials regard their original chemical characteristics as well as their capability for surface functionalization. The large specific surface area of 2D planar structured graphene allows loading or interacting with various chemical

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compounds and biological species via either covalent bonds or noncovalent interactions for tissue engineering applications. Both functionalized and unmodified graphene and GO can augment stem cell osteogenesis for bone tissue applications. Kumar et al. studied the performance of graphene nanoparticles with various surface chemical moieties in promoting stem cell osteogenesis. They demonstrated the amine-functionalized GO (AGO) exhibited the most significant effect in augmenting hMSC proliferation and osteogenesis, which was attributed to the synergistic effect of oxygen-containing functional groups and amine groups on AGO enabling a favorable stem cell response [87]. GO-doped PLGA nanofiber scaffolds have been proved to promote osteogenic differentiation of MSCs, mainly because the presence of GO not only enhanced the hydrophilic performance and protein adsorption ability, but also accelerated hMSC adhesion and proliferation [88]. Graphene could also remarkably accelerate osteogenic differentiation without commonly used additional bone growth factors. This might be due to the fact that graphene can increase calcium deposit or local dexamethasone concentration via π π stacking between the aromatic rings in the biomolecules and the graphene basal plane [46,89,90]. The chemical properties of graphene-based materials are also useful for their applications in the neural engineering. For example, coating poly-L-lactide nanofibers with GO rendered the scaffold with better hydrophilicity and could enhance cell materials interaction, and such a scaffold greatly improved the proliferation and differentiation of pheochromocytoma cells and neurite growth [91,92]. Weaver et al. introduced a nanocomposite composed of GO and a conducting PEDOT to study NSC differentiation. They demonstrated that the presence of GO improved the adsorption of proteins from the media, encouraging a high rate of proliferation and differentiation of NSCs. In addition, the free carboxylic acid group on GO facilitated covalent immobilization of biomolecular inducers on the surface, contributing to preferential differentiation into neuronal or oligodendrocyte lineage, respectively [93]. Laminin-attached graphene provided more favorable microenvironments for hNSC attachment and differentiation more toward neurons rather than glial cells. The mechanism might have resulted from significant upregulation of laminin-related receptors in hNSCs ECM on graphene [72]. Fluorinated graphene (FG) sheets have been used as the scaffold for stem cell growth. Wang et al. demonstrated that the strong polarization effect of the carbon fluorine bond might facilitate cell alignment and nucleus elongation through electrostatic induction at the interface of cell FG. Morphological changes in terms of cytoskeletal and nuclear alignment promoted the differentiation of MSCs toward the neuronal lineage [94]. Various biomolecule-absorbed GO flakes have been used in cardiac repair. After absorbing ECM proteins, GO could form a complex with MSCs and preserved the cell ECM interactions that could prevent the death of the cells caused by the generation of reactive oxygen species. Thus, GO flakes as a cellular adhesive could improve the therapeutic efficacy of MSC implantation, which has emerged as a potential therapy for MI [95]. Similarly, the ECM protein fibronectin-adsorbed rGO flakes enhanced the cell ECM interactions and expression of paracrine factor that further upregulated a gap junction protein,

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tion

MSC

injec

Infarct region MSC or MSC spheroid

MSC-RGO hybrid spheroid

RGO flake FN FN–adsorbed RGO flake

Cell–cell interaction

Cell–ECM interaction: Enhanced expression of Electroconductivity of RGO: paracrine factors and C×43 Enhanced C×43 expression

FIGURE 7–4 Schematic illustration of the effects of rGO flake incorporation in MSC spheroids on cardiac repair in MSC therapy for the treatment of MI. MI, Myocardial infarction; MSC, mesenchymal stem cell; rGO, reduced graphene oxide. Reprinted with permission from J. Park, Y.S. Kim, S. Ryu, W.S. Kang, S. Park, J. Han, et al., Graphene potentiates the myocardial repair efficacy of mesenchymal stem cells by stimulating the expression of angiogenic growth factors and gap junction protein, Adv. Funct. Mater. 25 (2015) 2590 2600.

leading to improvement of the cardiac repair efficiency (Fig. 7 4) [82]. Paul et al. reported GO could be ionically bonded to cationic polyethylenimine, acting as a gene-delivery agent with high efficiency. After being injected, GO carrying vascular endothelial growth factor facilitated specific local myocardial neovascularization, reduced fibrosis, and improved cardiac function [96]. The chemical properties of graphene-based materials have also been used in the skin and tissue engineering. Similar to the above discussion, the incorporation of GO into PLGA/collagen hybrid fiber matrices obtained a more hydrophilic surface that provided a suitable microenvironment to significantly enhance the attachment and proliferation of skeletal myoblasts, indicating that GO-impregnated hybrid matrices had potent effects on the induction of spontaneous myogenesis [52]. It has also been reported that the different oxidation states of GO and few-layer graphene were responsible for their subtle but differential effects on HaCaT human skin keratinocytes [97]. Therefore, the chemical properties of graphene-based materials render a hydrophilic surface and provide a substrate to adsorb ECM biomolecules, both of which can greatly enhance the cell ECM interaction and promote adhesion, proliferation, and differentiation of cells on the substrate. Proper surface oxidation or functionalization of graphene-based materials brings about a specific interaction with cells, enabling corresponding tissue engineering applications.

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7.2.4 Other Properties and Applications In addition to the above three dominant properties, other unique properties of graphenebased materials, including surface morphology, impermeability, and photothermal effects, make them suitable for some specific applications in the tissue engineering field. Wrinkles and ripples exist universally in graphene-based structures [98]. Both wrinkles and ripples can increase the surface roughness of graphene-based materials, facilitating strong absorption of proteins and enhancement of cell growth and differentiation. Lu et al. prepared a self-supporting graphene hydrogel film as a platform for bone regeneration. They found graphene could effectively enhance the osteogenic differentiation, mainly due to the rough surface morphology combining mechanical properties of self-supporting graphene hydrogel [42], as rough and disordered surfaces have been reported to induce bone cell differentiation [99]. Kim et al. fabricated rGO-incorporated chitosan substrate to investigate the adhesion and differentiation of hMSCs. They concluded the asymmetrical nanotopology of rGO chitosan substrate made it suitable for the adhesion and proliferation of hMSCs and enhanced cell substrate interaction and cell cell contacts. They demonstrated that the rGO chitosan substrate could promote both the osteogenesis and neurogenesis of hMSCs under different culture conditions [100]. The impermeability of graphene allows graphene acting as a biocompatible anticorrosion coating to protect metallic biomedical devices that have been widely used in the tissue engineering field, such as bone implants. Graphene coating could enhance both the biocompatibility and hemocompatibility of implant materials [101,102]. Zhang and colleagues conducted both in vitro and in vivo experiments to demonstrate the use of graphene coating as an effective protection film under biological environments (Fig. 7 5) [103]. Their results open up the potential of applying graphene to protect metal devices in biomedical applications. GO/HA-coated Ti substrate has also been reported to exhibit high corrosion resistance with enhanced coating adhesion strength and superior cell viability [104]. The photothermal effect of GO can be used to fabricate a near-infrared (NIR) lighttriggered active scaffold for reversible cell capture and on-demand release, which may have future applications in tissue engineering and cell-based therapy. Li et al. fabricated such a scaffold by combining GO and thermo-responsive poly(N-isopropylacrylamide) (pNIPAAm). They demonstrated the 3D hybrid porous hydrogel could efficiently capture cells not only through the bioadhesive GO but could also release the cells upon an NIR light stimulus, realizing better dynamic control on cells than traditional passive cell depots [105]. The intrinsic wrinkles and ripples of graphene can increase the surface roughness of graphene-based composites, which is helpful for adsorbing biomolecules to induce the interactions between graphene and cells, leading to the enhancement of cellular adhesion, proliferation, and differentiation. The impermeability and photothermal property of graphene and GO enable them to be used either as a potential protective coating for metal implants or to fabricate a smart active scaffold to dynamically manipulate cells.

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FIGURE 7–5 Graphene as a protective coating in biological environments. (A) Schematic illustration of graphene as a protective layer on a metal surface. (B) Concentration of Cu21 ions in blood extracted from live rats. Concentrations of Cu21 ions from normal rats before implantation as control (black square), rats with implanted single-layer graphene (SLG)/Cu foils (blue triangles), and rats with implanted Cu foils (red dots) as a function of time after implantation. (C) Relative cell viability of bone cells incubated with SLG/Cu, bilayer graphene (BLG)/Cu, and bare Cu foil for 1 day. The control was the regular cell culture without the presence of any form of Cu foil. Reprinted with permission from H. Zhao, R. Ding, X. Zhao, Y. Li, L. Qu, H. Pei, et al., Graphene-based nanomaterials for drug and/or gene delivery, bioimaging, and tissue engineering, Drug Discov. Today 22 (2017) 1302 1317.

7.3 Conclusion In this chapter, we have reviewed various applications of graphene and GO in tissue engineering. Due to the unique 2D chemical structure, graphene-based materials have fascinating mechanical and electrical properties, versatile surface chemistry, and many other intrinsic properties including rough morphology, impermeability, and photothermal effects. These excellent properties render graphene and GO to be extensively applied in the field of tissue engineering. We summarize applications of graphene and GO in bone, neural, cardiac, skin, and muscle tissue engineering and regeneration, and discuss the underneath mechanisms for each application. We demonstrate at least one specific property that graphene and GO contribute to each mechanism. Although we organize this chapter based on the properties of graphene and GO and discuss the effect of each property on their applications, we want to clarify that in most case the functions of graphene-based materials in tissue engineering applications are attributed to the synergistic effect of two or even more properties. We hope this chapter not only provides an overview of tissue engineering applications of graphene and GO, but also, more importantly, gives an effective instruction to extend their biomedical applications in the future.

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References [1] H. Zhao, R. Ding, X. Zhao, Y. Li, L. Qu, H. Pei, et al., Graphene-based nanomaterials for drug and/or gene delivery, bioimaging, and tissue engineering, Drug Discov. Today 22 (2017) 1302 1317. [2] A.K. Geim, K.S. Novoselov, The rise of graphene, Nat. Mater. 6 (2007) 183 191. [3] Y. Zhu, S. Murali, W. Cai, X. Li, J.W. Suk, J.R. Potts, et al., Graphene and graphene oxide: synthesis, properties, and applications, Adv. Mater. 22 (2010) 3906 3924. [4] J.S. Bunch, A.M. van der Zande, S.S. Verbridge, I.W. Frank, D.M. Tanenbaum, J.M. Parpia, et al., Electromechanical resonators from graphene sheets, Science 315 (2007) 490 493. [5] F. Schedin, A.K. Geim, S.V. Morozov, E.W. Hill, P. Blake, M.I. Katsnelson, et al., Detection of individual gas molecules adsorbed on graphene, Nat. Mater. 6 (2007) 652 655. [6] X. Du, I. Skachko, A. Barker, E.Y. Andrei, Approaching ballistic transport in suspended graphene, Nat. Nanotechnol. 3 (2008) 491 495. [7] G. Eda, G. Fanchini, M. Chhowalla, Large-area ultrathin films of reduced graphene oxide as a transparent and flexible electronic material, Nat. Nanotechnol. 3 (2008) 270 274. [8] A.K. Geim, Graphene: status and prospects, Science 324 (2009) 1530 1534. [9] Z. Liu, J.T. Robinson, X. Sun, H. Dai, PEGylated nanographene oxide for delivery of water-insoluble cancer drugs, J. Am. Chem. Soc. 130 (2008) 10876 10877. [10] X. Sun, Z. Liu, K. Welsher, J.T. Robinson, A. Goodwin, S. Zaric, et al., Nano-graphene oxide for cellular imaging and drug delivery, Nano Res. 1 (2008) 203 212. [11] J. Liu, L. Cui, D. Losic, Graphene and graphene oxide as new nanocarriers for drug delivery applications, Acta Biomater. 9 (2013) 9243 9257. [12] M. Vincent, I. de Lázaro, K. Kostarelos, Graphene materials as 2D non-viral gene transfer vector platforms, Gene Ther. 24 (2016) 123 132. [13] J. Lin, X. Chen, P. Huang, Graphene-based nanomaterials for bioimaging, Adv. Drug Deliv. Rev. 105 (2016) 242 254. [14] J. Li, W. Zhang, T.-F. Chung, M.N. Slipchenko, Y.P. Chen, J.-X. Cheng, et al., Highly sensitive transient absorption imaging of graphene and graphene oxide in living cells and circulating blood, Sci. Rep. 5 (2015) 12394. [15] C.I.L. Justino, A.R. Gomes, A.C. Freitas, A.C. Duarte, T.A.P. Rocha-Santos, Graphene based sensors and biosensors, TrAC Trend Anal. Chem. 91 (2017) 53 66. [16] W. Hu, C. Peng, W. Luo, M. Lv, X. Li, D. Li, et al., Graphene-based antibacterial paper, ACS Nano 4 (2010) 4317 4323. [17] N. Li, X. Zhang, Q. Song, R. Su, Q. Zhang, T. Kong, et al., The promotion of neurite sprouting and outgrowth of mouse hippocampal cells in culture by graphene substrates, Biomaterials 32 (2011) 9374 9382. [18] N. Shadjou, M. Hasanzadeh, B. Khalilzadeh, Graphene based scaffolds on bone tissue engineering, Bioengineered 9 (2018) 38 47. [19] S.R. Shin, Y.-C. Li, H.L. Jang, P. Khoshakhlagh, M. Akbari, A. Nasajpour, et al., Graphene-based materials for tissue engineering, Adv. Drug Deliv. Rev. 105 (2016) 255 274. [20] Y. Yang, A.M. Asiri, Z. Tang, D. Du, Y. Lin, Graphene based materials for biomedical applications, Mater. Today 16 (2013) 365 373. [21] J. Kim, K.S. Choi, Y. Kim, K.-T. Lim, H. Seonwoo, Y. Park, et al., Bioactive effects of graphene oxide cell culture substratum on structure and function of human adipose-derived stem cells, J. Biomed. Mater. Res. A 101 (2013) 3520 3530.

Chapter 7 • Graphene and Graphene Oxide for Tissue Engineering and Regeneration

181

[22] G. Reina, J.M. González-Domínguez, A. Criado, E. Vázquez, A. Bianco, M. Prato, Promises, facts and challenges for graphene in biomedical applications, Chem. Soc. Rev 46 (2017) 4400 4416. [23] Y. Ma, J. Huang, H. Shen, M. Zhang, S. Song, Z. Zhang, Graphene for biomedical applications, in: M. Zhang, R.R. Naik, L. Dai (Eds.), Carbon Nanomaterials for Biomedical Applications, Springer International Publishing, Cham, 2016, pp. 241 267. [24] F.G. Heineken, R. Skalak, Tissue engineering: a brief overview, J. Biomech. Eng. 113 (1991) 111 112. [25] R. Langer, J. Vacanti, Tissue engineering, Science 260 (1993) 920 926. [26] M.D. Stoller, S. Park, Y. Zhu, J. An, R.S. Ruoff, Graphene-based ultracapacitors, Nano Lett. 8 (2008) 3498 3502. [27] C. Lee, X. Wei, J.W. Kysar, J. Hone, Measurement of the elastic properties and intrinsic strength of monolayer graphene, Science 321 (2008) 385 388. [28] A.A. Balandin, S. Ghosh, W. Bao, I. Calizo, D. Teweldebrhan, F. Miao, et al., Superior thermal conductivity of single-layer graphene, Nano Lett. 8 (2008) 902 907. [29] W. Gao, L.B. Alemany, L. Ci, P.M. Ajayan, New insights into the structure and reduction of graphite oxide, Nat. Chem. 1 (2009) 403 408. [30] K.I. Bolotin, K.J. Sikes, Z. Jiang, M. Klima, G. Fudenberg, J. Hone, et al., Ultrahigh electron mobility in suspended graphene, Solid State Commun. 146 (2008) 351 355. [31] S.V. Morozov, K.S. Novoselov, M.I. Katsnelson, F. Schedin, D.C. Elias, J.A. Jaszczak, et al., Giant intrinsic carrier mobilities in graphene and its bilayer, Phys. Rev. Lett. 100 (2008) 016602. [32] S. Viswanathan, T.N. Narayanan, K. Aran, K.D. Fink, J. Paredes, P.M. Ajayan, et al., Graphene protein field effect biosensors: glucose sensing, Mater. Today 18 (2015) 513 522. [33] K.P. Loh, Q. Bao, P.K. Ang, J. Yang, The chemistry of graphene, J. Mater. Chem. 20 (2010) 2277 2289. [34] V. Georgakilas, J.N. Tiwari, K.C. Kemp, J.A. Perman, A.B. Bourlinos, K.S. Kim, et al., Noncovalent functionalization of graphene and graphene oxide for energy materials, biosensing, catalytic, and biomedical applications, Chem. Rev. 116 (2016) 5464 5519. [35] A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, Matrix elasticity directs stem cell lineage specification, Cell 126 (2006) 677 689. [36] J. Venkatesan, R. Pallela, S.-K. Kim, Applications of carbon nanomaterials in bone tissue engineering, J. Biomed. Nanotechnol. 10 (2014) 3105 3123. [37] Q. Wang, Y. Chu, J. He, W. Shao, Y. Zhou, K. Qi, et al., A graded graphene oxide-hydroxyapatite/silk fibroin biomimetic scaffold for bone tissue engineering, Mater. Sci. Eng. C Mater. Biol. Appl. 80 (2017) 232 242. [38] C. Fu, H. Bai, J. Zhu, Z. Niu, Y. Wang, J. Li, et al., Enhanced cell proliferation and osteogenic differentiation in electrospun PLGA/hydroxyapatite nanofibre scaffolds incorporated with graphene oxide, PLoS One 12 (2017) e0188352. [39] X. Xie, K. Hu, D. Fang, L. Shang, S.D. Tran, M. Cerruti, Graphene and hydroxyapatite self-assemble into homogeneous, free standing nanocomposite hydrogels for bone tissue engineering, Nanoscale 7 (2015) 7992 8002. [40] M. Nair, D. Nancy, A.G. Krishnan, G.S. Anjusree, S. Vadukumpully, S.V. Nair, Graphene oxide nanoflakes incorporated gelatin-hydroxyapatite scaffolds enhance osteogenic differentiation of human mesenchymal stem cells, Nanotechnology 26 (2015) 161001. [41] R. Rajesh, Y.D. Ravichandran, Development of new graphene oxide incorporated tricomponent scaffolds with polysaccharides and hydroxyapatite and study of their osteoconductivity on MG-63 cell line for bone tissue engineering, RSC Adv. 5 (2015) 41135 41143.

182

THERANOSTIC BIONANOMATERIALS

[42] J. Lu, Y.-S. He, C. Cheng, Y. Wang, L. Qiu, D. Li, et al., Self-supporting graphene hydrogel film as an experimental platform to evaluate the potential of graphene for bone regeneration, Adv. Funct. Mater. 23 (2013) 3494 3502. [43] E. Nishida, H. Miyaji, H. Takita, I. Kanayama, M. Tsuji, T. Akasaka, et al., Graphene oxide coating facilitates the bioactivity of scaffold material for tissue engineering, JPN J. Appl. Phys. 53 (2014). [44] L. Wang, C. Lu, Y. Li, F. Wu, B. Zhao, X. Dong, Green fabrication of porous silk fibroin/graphene oxide hybrid scaffolds for bone tissue engineering, RSC Adv. 5 (2015) 78660 78668. [45] G. Lalwani, A.M. Henslee, B. Farshid, L. Lin, F.K. Kasper, Y.X. Qin, et al., Two-dimensional nanostructure-reinforced biodegradable polymeric nanocomposites for bone tissue engineering, Biomacromolecules 14 (2013) 900 909. [46] T.R. Nayak, H. Andersen, V.S. Makam, C. Khaw, S. Bae, X. Xu, et al., Graphene for controlled and accelerated osteogenic differentiation of human mesenchymal stem cells, ACS Nano 5 (2011) 4670 4678. [47] C. Gao, T. Liu, C. Shuai, S. Peng, Enhancement mechanisms of graphene in nano-58S bioactive glass scaffold: mechanical and biological performance, Sci. Rep. 4 (2014) 4712. [48] H.K.F. Cheng, N.G. Sahoo, Y.P. Tan, Y. Pan, H. Bao, L. Li, et al., Poly(vinyl alcohol) nanocomposites filled with poly(vinyl alcohol)-grafted graphene oxide, ACS Appl. Mater. Interfaces 4 (2012) 2387 2394. [49] Y. Du, J. Ge, Y. Li, P.X. Ma, B. Lei, Biomimetic elastomeric, conductive and biodegradable polycitratebased nanocomposites for guiding myogenic differentiation and skeletal muscle regeneration, Biomaterials 157 (2018) 40 50. [50] C. Cha, S.R. Shin, X. Gao, N. Annabi, M.R. Dokmeci, X. Tang, et al., Controlling mechanical properties of cell-laden hydrogels by covalent incorporation of graphene oxide, Small 10 (3) (2014) 514 523. [51] H. Jo, M. Sim, S. Kim, S. Yang, Y. Yoo, J.-H. Park, et al., Electrically conductive graphene/polyacrylamide hydrogels produced by mild chemical reduction for enhanced myoblast growth and differentiation, Acta Biomater. 48 (2017) 100 109. [52] Y.C. Shin, J.H. Lee, L. Jin, M.J. Kim, Y.-J. Kim, J.K. Hyun, et al., Stimulated myoblast differentiation on graphene oxide-impregnated PLGA-collagen hybrid fibre matrices, J. Nanobiotechnol. 13 (2015) 21. [53] A. Patel, Y. Xue, S. Mukundan, L.C. Rohan, V. Sant, D.B. Stolz, et al., Cell-instructive graphene-containing nanocomposites induce multinucleated myotube formation, Ann. Biomed. Eng. 44 (2016) 2036 2048. [54] G.W. Rogers, J.Z. Liu, Monolayer graphene oxide as a building block for artificial muscles, Appl. Phys. Lett. 102 (2013) 021903. [55] J. Zang, S. Ryu, N. Pugno, Q. Wang, Q. Tu, M.J. Buehler, et al., Multifunctionality and control of the crumpling and unfolding of large-area graphene, Nat. Mater. 12 (2013) 321 325. [56] J. Kim, J.-H. Jeon, H.-J. Kim, H. Lim, I.-K. Oh, Highly sustainable electroactive artificial muscle with graphene paper electrodes, in: Advanced Intelligent Mechatronics (AIM), 2014 IEEE/ASME International Conference on, IEEE, 2014, . 1442 1445. [57] M. Kotal, J. Kim, K.J. Kim, I.K. Oh, Sulfur and nitrogen co-doped graphene electrodes for high-performance ionic artificial muscles, Adv. Mater 28 (2016) 1610 1615. [58] S.J. Kim, K.W. Cho, H.R. Cho, L. Wang, S.Y. Park, S.E. Lee, et al., Stretchable and transparent biointerface using cell-sheet graphene hybrid for electrophysiology and therapy of skeletal muscle, Adv. Funct. Mater. 26 (2016) 3207 3217. [59] B. Nyambat, C.-H. Chen, P.-C. Wong, C.-W. Chiang, M.K. Satapathy, E.-Y. Chuang, Genipin-crosslinked adipose stem cell derived extracellular matrix-nano graphene oxide composite sponge for skin tissue engineering, J. Mater. Chem. B 6 (2018) 979 990. [60] R. Deepachitra, V. Ramnath, T.P. Sastry, Graphene oxide incorporated collagen fibrin biofilm as a wound dressing material, RSC Adv. 4 (2014) 62717 62727.

Chapter 7 • Graphene and Graphene Oxide for Tissue Engineering and Regeneration

183

[61] Z. Li, H. Wang, B. Yang, Y. Sun, R. Huo, Three-dimensional graphene foams loaded with bone marrow derived mesenchymal stem cells promote skin wound healing with reduced scarring, Mater. Sci. Eng. C 57 (2015) 181 188. [62] C. Hou, T. Huang, H. Wang, H. Yu, Q. Zhang, Y. Li, A strong and stretchable self-healing film with selfactivated pressure sensitivity for potential artificial skin applications, Sci. Rep. 3 (2013) 3138. [63] R. Bao, B.Y. Tan, S. Liang, N. Zhang, W. Wang, W.G. Liu, A pi-pi conjugation-containing soft and conductive injectable polymer hydrogel highly efficiently rebuilds cardiac function after myocardial infarction, Biomaterials 122 (2017) 63 71. [64] A.J. Ryan, C.J. Kearney, N. Shen, U. Khan, A.G. Kelly, C. Probst, et al., Electroconductive biohybrid collagen/pristine graphene composite biomaterials with enhanced biological activity, Adv. Mater. 30 (2018) 1706442. [65] J. Zhou, X. Yang, W. Liu, C. Wang, Y. Shen, F. Zhang, et al., Injectable OPF/graphene oxide hydrogels provide mechanical support and enhance cell electrical signaling after implantation into myocardial infarct, Theranostics 8 (2018) 3317 3330. [66] S.R. Shin, C. Zihlmann, M. Akbari, P. Assawes, L. Cheung, K. Zhang, et al., Reduced graphene oxideGelMA hybrid hydrogels as scaffolds for cardiac tissue engineering, Small 12 (2016) 3677 3689. [67] N. Annabi, S.R. Shin, A. Tamayol, M. Miscuglio, M.A. Bakooshli, A. Assmann, et al., Highly elastic and conductive human-based protein hybrid hydrogels, Adv. Mater. 28 (2016) 40 49. [68] S. Ahadian, Y. Zhou, S. Yamada, M. Estili, X. Liang, K. Nakajima, et al., Graphene induces spontaneous cardiac differentiation in embryoid bodies, Nanoscale 8 (2016) 7075 7084. [69] Z.Q. Feng, T. Wang, B. Zhao, J. Li, L. Jin, Soft graphene nanofibers designed for the acceleration of nerve growth and development, Adv. Mater. 27 (2015) 6462 6468. [70] S. Sánchez-González, N. Diban, A. Urtiaga, Hydrolytic degradation and mechanical stability of poly (ε-caprolactone)/reduced graphene oxide membranes as scaffolds for in vitro neural tissue regeneration, Membranes 8 (2018) 12. [71] D.W. Park, A.A. Schendel, S. Mikael, S.K. Brodnick, T.J. Richner, J.P. Ness, et al., Graphene-based carbon-layered electrode array technology for neural imaging and optogenetic applications, Nat. Commun. 5 (2014) 5258. [72] S.Y. Park, J. Park, S.H. Sim, M.G. Sung, K.S. Kim, B.H. Hong, et al., Enhanced differentiation of human neural stem cells into neurons on graphene, Adv. Mater. 23 (2011) H263 H267. [73] R. Guo, S. Zhang, M. Xiao, F. Qian, Z. He, D. Li, et al., Accelerating bioelectric functional development of neural stem cells by graphene coupling: implications for neural interfacing with conductive materials, Biomaterials 106 (2016) 193 204. [74] W. Guo, X. Zhang, X. Yu, S. Wang, J. Qiu, W. Tang, et al., Self-powered electrical stimulation for enhancing neural differentiation of mesenchymal stem cells on graphene-poly(3,4-ethylenedioxythiophene) hybrid microfibers, ACS Nano 10 (2016) 5086 5095. [75] N. Li, Q. Zhang, S. Gao, Q. Song, R. Huang, L. Wang, et al., Three-dimensional graphene foam as a biocompatible and conductive scaffold for neural stem cells, Sci. Rep. 3 (2013) 1604. [76] K. Yang, Y. Li, X. Tan, R. Peng, Z. Liu, Behavior and toxicity of graphene and its functionalized derivatives in biological systems, Small 9 (2013) 1492 1503. [77] M. Lorenzoni, F. Brandi, S. Dante, A. Giugni, B. Torre, Simple and effective graphene laser processing for neuron patterning application, Sci. Rep. 3 (2013) 1954. [78] X. Liu, A.L. Miller Ii, S. Park, B.E. Waletzki, A. Terzic, M.J. Yaszemski, et al., Covalent crosslinking of graphene oxide and carbon nanotube into hydrogels enhances nerve cell responses, J. Mater. Chem. B 4 (2016) 6930 6941. [79] G.A. Silva, Neuroscience nanotechnology: progress, opportunities and challenges, Nat. Rev. Neurosci. 7 (2006) 65 74.

184

THERANOSTIC BIONANOMATERIALS

[80] M. Tang, Q. Song, N. Li, Z. Jiang, R. Huang, G. Cheng, Enhancement of electrical signaling in neural networks on graphene films, Biomaterials 34 (2013) 6402 6411. [81] T.-C. Liu, M.-C. Chuang, C.-Y. Chu, W.-C. Huang, H.-Y. Lai, C.-T. Wang, et al., Implantable graphenebased neural electrode interfaces for electrophysiology and neurochemistry in in vivo hyperacute stroke model, ACS Appl. Mater. Interfaces 8 (2015) 187 196. [82] J. Park, Y.S. Kim, S. Ryu, W.S. Kang, S. Park, J. Han, et al., Graphene potentiates the myocardial repair efficacy of mesenchymal stem cells by stimulating the expression of angiogenic growth factors and gap junction protein, Adv. Funct. Mater. 25 (2015) 2590 2600. [83] A.S.T. Smith, H. Yoo, H. Yi, E.H. Ahn, J.H. Lee, G. Shao, et al., Micro- and nano-patterned conductive graphene-PEG hybrid scaffolds for cardiac tissue engineering, Chem. Commun. 53 (2017) 7412 7415. [84] P. Bajaj, J.A. Rivera, D. Marchwiany, V. Solovyeva, R. Bashir, Graphene-based patterning and differentiation of C2C12 myoblasts, Adv. Healthc. Mater. 3 (2014) 995 1000. [85] S. Ahadian, J. Ramón-Azcón, H. Chang, X. Liang, H. Kaji, H. Shiku, et al., Electrically regulated differentiation of skeletal muscle cells on ultrathin graphene-based films, RSC Adv. 4 (2014) 9534 9541. [86] C. Hou, H. Wang, Q. Zhang, Y. Li, M. Zhu, Highly conductive, flexible, and compressible all-graphene passive electronic skin for sensing human touch, Adv. Mater. 26 (2014) 5018 5024. [87] S. Kumar, S. Raj, E. Kolanthai, A.K. Sood, S. Sampath, K. Chatterjee, Chemical functionalization of graphene to augment stem cell osteogenesis and inhibit biofilm formation on polymer composites for orthopedic applications, ACS Appl. Mater. Interfaces 7 (2015) 3237 3252. [88] Y. Luo, H. Shen, Y. Fang, Y. Cao, J. Huang, M. Zhang, et al., Enhanced proliferation and osteogenic differentiation of mesenchymal stem cells on graphene oxide-incorporated electrospun poly(lactic-coglycolic acid) nanofibrous mats, ACS Appl. Mater. Interfaces 7 (2015) 6331 6339. [89] O. Akhavan, E. Ghaderi, M. Shahsavar, Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells, Carbon 59 (2013) 200 211. [90] W.C. Lee, C.H.Y.X. Lim, H. Shi, L.A.L. Tang, Y. Wang, C.T. Lim, et al., Origin of enhanced stem cell growth and differentiation on graphene and graphene oxide, ACS Nano 5 (2011) 7334 7341. [91] J. Wu, L. Xie, W.Z.Y. Lin, Q. Chen, Biomimetic nanofibrous scaffolds for neural tissue engineering and drug development, Drug Discov. Today 22 (2017) 1375 1384. [92] K. Zhang, H. Zheng, S. Liang, C. Gao, Aligned PLLA nanofibrous scaffolds coated with graphene oxide for promoting neural cell growth, Acta Biomater. 37 (2016) 131 142. [93] C.L. Weaver, X.T. Cui, Directed neural stem cell differentiation with a functionalized graphene oxide nanocomposite, Adv. Healthc. Mater. 4 (2015) 1408 1416. [94] Y. Wang, W.C. Lee, K.K. Manga, P.K. Ang, J. Lu, Y.P. Liu, et al., Fluorinated graphene for promoting neuro-induction of stem cells, Adv. Mater. 24 (2012) 4285 4290. [95] J. Park, B. Kim, J. Han, J. Oh, S. Park, S. Ryu, et al., Graphene oxide flakes as a cellular adhesive: prevention of reactive oxygen species mediated death of implanted cells for cardiac repair, ACS Nano 9 (2015) 4987 4999. [96] A. Paul, A. Hasan, H. Al Kindi, A.K. Gaharwar, V.T.S. Rao, M. Nikkhah, et al., Injectable graphene oxide/ hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair, ACS Nano 8 (2014) 8050 8062. [97] J. Frontinan-Rubio, M.V. Gomez, C. Martin, J.M. Gonzalez-Dominguez, M. Duran-Prado, E. Vazquez, Differential effects of graphene materials on the metabolism and function of human skin cells, Nanoscale 10 (2018) 11604 11615. [98] L. Yang, T. Niu, H. Zhang, W. Xu, M. Zou, L. Xu, et al., Self-assembly of suspended graphene wrinkles with high pre-tension and elastic property, 2D Mater. 4 (2017) 041001.

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[99] M.J. Dalby, N. Gadegaard, R. Tare, A. Andar, M.O. Riehle, P. Herzyk, et al., The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder, Nat. Mater. 6 (2007) 997 1003. [100] J. Kim, Y.-R. Kim, Y. Kim, K.T. Lim, H. Seonwoo, S. Park, et al., Graphene-incorporated chitosan substrata for adhesion and differentiation of human mesenchymal stem cells, J. Mater. Chem. B 1 (2013) 933 938. [101] R. Podila, T. Moore, F. Alexis, A. Rao, Graphene coatings for enhanced hemo-compatibility of nitinol stents, RSC Adv. 3 (2013) 1660 1665. [102] R. Podila, T. Moore, F. Alexis, A. Rao, Graphene coatings for biomedical implants, J. Vis. Exp (2013) e50276. [103] W. Zhang, S. Lee, K.L. McNear, T.F. Chung, S. Lee, K. Lee, et al., Use of graphene as protection film in biological environments, Sci. Rep. 4 (2014) 4097. [104] M. Li, Q. Liu, Z. Jia, X. Xu, Y. Cheng, Y. Zheng, et al., Graphene oxide/hydroxyapatite composite coatings fabricated by electrophoretic nanotechnology for biological applications, Carbon 67 (2014) 185 197. [105] W. Li, J. Wang, J. Ren, X. Qu, 3D graphene oxide polymer hydrogel: near-infrared light-triggered active scaffold for reversible cell capture and on-demand release, Adv. Mater. 25 (2013) 6737 6743.

8 Nanomaterial Design and Tests for Neural Tissue Engineering Jiajia Xue THE W ALLACE H. COULTER D EPARTMENT OF B IOMEDICAL E NGINEERING, GEORGIA INSTITUTE OF TECHNOLOGY AND EMORY UNIVERSITY, ATLANTA, GA, UNITED STATES

The nervous system is a network of neural cells that transmit related signals to synchronize the brain and parts of the body. The nervous system can be divided into two main regions in the human body: the central nervous system (CNS) (including the brain and the spinal cord) and the peripheral nervous system (PNS) (including the spinal and autonomic nerves). Nerves emerge from the CNS through the skull and vertebral column, using the PNS to carry information to the rest of the body [1]. Neurons are electrically excitable cells that maintain a voltage gradient across their membranes by using ion pumps that generate concentration gradient differences of ions such as potassium, calcium, sodium, and chloride [2]. A nerve injury that has occurred in the CNS or PNS is a major problem that can lead to serious disability, and affects millions of people around the world annually. Growing efforts are dedicated to the development of effective treatment for nerve injury, to improve tissue regeneration and functional recovery. Neural tissue engineering involving the integration of scaffolding materials, cells, and/or biological factors represents a promising strategy to repair nerve injuries, which might be eventually translated to patients for improving the clinical outcome [3]. It is desirable to design tissue-engineered scaffolding materials mimicking the native environment of the tissues, which can provide a suitable combination of mechanical support, topographic guidance, biochemical instruction, and electrical simulation to the neurons and the related cells. In native tissues, the cells directly interact with (and create) nanostructured extracellular matrix (ECM). To this end, nanomaterials with biomimetic features and excellent physiochemical properties play a key role in stimulating and guiding neural regeneration [4,5]. For achieving an optimal outcome in repairing nerve injury, various types of nanomaterials have been designed and developed with engineered composition, architecture, and functional properties to emulate the native ECM as closely as possible. In this chapter, we discuss the basic requirements and strategies of the design and testing of nanomaterials for neural tissue engineering by focusing on their applications in the repair of the spinal cord injury (SCI) and the peripheral nerve injury (PNI).

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8.1 Design of Nanomaterials for Neural Tissue Engineering The ideal nanomaterials for neural tissue engineering applications should have excellent cytocompatibility, mechanical strength, electrical property, and suitable degradability. The main requirement for nanomaterials is biocompatibility to avoid inhibition of cell growth and the occurrence of inflammation or infection that can greatly inhibit nerve regeneration [4,6]. Sufficient mechanical strength is required for the nanomaterials to last a long enough period to physically supporting the newly formed nerve tissue. The nanomaterials must also be fabricated into a three-dimensional (3D) porous structure for new nerve tissue formation, along with the ability to transport nutrients and waste. In addition, superior electrical properties of the nanomaterials are desirable to help stimulate and control neuron behavior under electrical stimulation, thus, more effectively guiding neural tissue repair. Furthermore, the nanomaterials should be able to degrade in the body along with the regeneration process to leave space for the formation of new nerve tissue, avoiding second surgery to remove the scaffolding nanomaterials. In addition to these basic requirements, from the perspective of the functional point, it is of critical importance for the scaffolding nanomaterials to mimic the native 3D nanofibrous structure of the ECM as close as possible in terms of composition, architecture, and properties. To date, submicron and nanoscale fibrous scaffolds fabricated by electrospinning and self-assembly can be potential scaffold candidates for neural tissue engineering. Electrospinning produces fibers with diameters ranging from tens of nanometers to several micrometers, which can be used to construct fibrous nerve conduits and introduced at lesion sites by implantation to repair both the SCI and PNI. Self-assembly fibers have diameters of tens of nanometers and can be injected for SCI repair and combined with a tube for PNI repair. Both functional nanofibrous scaffolds would enhance neurite extension and axon regrowth, serving as powerful tools for neural tissue engineering. Various natural and synthetic materials have been adopted to construct the nerve grafts to repair severely damaged nerves by bridging nerve gaps and guiding neurite outgrowth [6]. When characterizing the effect of the scaffolds for the development of successful clinical approaches, the in vitro models act as the first step in the pipeline toward in vivo studies followed by clinical trials [5,7]. Depending on the type of the injury, the in vitro cell model and test method are different. Followed by the in vitro characterization, by optimizing the properties of the scaffold, in vivo animal models are established to examine the functional recovery of the injured nerve under the assistance of the scaffolds. The in vivo outcome will further guide the design and modification of the scaffold to achieve further improvement.

8.2 Nanomaterials for the Repair of Spinal Cord Injury Severe SCI results in paraplegia or quadriplegia and there is not yet an effective therapeutic approach for complete recovery. During the first few weeks after the injury, the

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microenvironment of the spinal cord changes considerably and an astrocytic scar is often formed, causing raised neuron and oligodendrocyte cell death and the upregulation of inhibitory factors of the axonal elongation [8]. In this case, the axon re-regrowth is restricted by a combination of the intrinsic and extrinsic factors: (1) the reduced capacity of the neuronal cell body to sustain a growth activated state, (2) the formation of a glial scar, and (3) the deposition of factors inhibitory to axonal growth at the injury site; for example, chondroitin sulfate proteoglycans and myelin-associated inhibitors of regeneration [9]. In order to enhance or enable the regeneration of spared axons and thus to reinstitute conduction in injured spinal cords with the goal of promoting functional recovery, the design of biomimetic nanofibrous scaffold has remained an area of intensive research.

8.2.1 Design of the Nanomaterials for Repairing the Spinal Cord Injury To repair the SCI injury, the scaffold is usually implanted at the lesion epicenter to provide a substrate for axonal growth and guidance. The scaffolding material can be a 3D structured bridge that incorporates guidance conduits or channels to direct the axonal outgrowth through the damaged region of the spinal cord [9 11]. Nanofibers fabricated by electrospinning or self-assembly represent one of the most promising nanomaterials to construct the guidance conduits or the channels. The scaffolding material can also be an injectable nanocompound that can fill the SCI cystic cavity and increase axon outgrowth into or adjacent to the scaffold [12]. Hydrogels represent one of the most promising materials for the injectable scaffold. The performance of the scaffold is firstly characterized in vitro by culturing with the neurons and the glial cells and/or the neural stem cells (NSCs) to examine the neuron regeneration and/or cell growth. Then, an in vivo model with a SCI in the mouse and/or rat is typically utilized to examine the performance of the scaffold, and the preclinical study is usually performed in a large animal model. Various types of natural and synthetic materials have been electrospun into nanofibers, which are feasible for SCI treatment. For example, based on nanofibers made of a blend of poly(l-lactide-co-ε-caprolactone) [P(LLA-CL)] and collagen, the NSCs were transdifferentiated into neuronal cells [13]. The presence of collagen significantly enhanced the attachment and differentiation of the NSCs compared with pure P(LLA-CL) nanofibers, which was attributed to the NSCs recognizing specific regions of collagen molecules. The surface topographical cues provided by the scaffolding materials play important roles in controlling cell behavior. For example, the uniaxially aligned nanofibers can guide the directional migration of cells and the preferential extension of neurites. In one study, from a rat hemisection model, the conduits made of aligned collagen nanofibers could diminish the accumulation of astrocytes at the lesion site, providing a permissive environment to allow for the cellular infiltration and to support the sprouting of neural fibers [14]. Along with the appropriate topographical structures and chemical cues, scaffolds that present proper mechanical characteristics provide another tool for repairing spinal cord nerve injury. For example, hydrogel can be combined with electrospun nanofibers for improving regeneration of the spinal cord. In one study, a 3D aligned fibrin nanofiber

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hydrogel with oriented structure and low elasticity (c.1.5 kPa) was developed via electrospinning and a molecular self-assembly process in a rotating collector within a liquid bath [15,16]. Fig. 8 1A and B shows the macrophoto and scanning electron microscopy (SEM) image of the scaffold, respectively [16]. The “nanofiber hydrogel” could mimic both the soft

FIGURE 8–1 (A) Macrophoto and (B) SEM image showing the network architecture of the aligned fibrin nanofiber hydrogel. (C and D) Immunofluorescence images showing the extension of neurites from DRG on the aligned fibrin nanofiber hydrogel. The distance of neurite extension was 1.96 mm in 3 days. (E) Immunofluorescence staining images of the longitudinal tissue section from the T8 T10 spinal cord segment at 4 weeks after implantation with aligned fibrin nanofiber hydrogel in the rat T9 dorsal hemisection lesion site. Inset shows the schematic diagram of the hemisection model. (F and G) H&E staining images of the coronal sections showing the structure of the spinal cord tissue after 12 weeks implantation of the (F) aligned nanofiber fibrin hydrogel and (G) saline, respectively, in a SCI dog model. DRG, Dorsal root ganglia; H&E, hematoxylin and eosin; SCI, spinal cord injury; SEM, scanning electron microscopy. (A, B, F, and G) Reprinted with permission from Z. Zhang, S. Yao, S. Xie, X. Wang, F. Chang, J. Luo, et al., Effect of hierarchically aligned fibrin hydrogel in regeneration of spinal cord injury demonstrated by tractography: a pilot study, Sci. Rep. 7 (2017) 40017. © 2017, Nature Publishing Group. (C E) Reprinted with permission from S. Yao, X. Liu, S. Yu, X. Wang, S. Zhang, Q. Wu, et al., Co-effects of matrix low elasticity and aligned topography on stem cell neurogenic differentiation and rapid neurite outgrowth, Nanoscale 8 (19) (2016) 10252 10265. © 2011, Royal Society of Chemistry.

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and oriented features of native nerve tissue [15]. In comparison with the random fibrin nanofiber hydrogel and rigid tissue culture plate, the aligned fibrin nanofiber hydrogel showed promoting effects on directing stem cell neurogenic differentiation and rapid neurite outgrowth in vitro. As shown in Fig. 8 1C, the aligned fibrin nanofiber hydrogel induced dorsal root ganglia (DRG) neurons to rapidly project numerous long neurites longitudinally along the fibers, without supplementation of neurotrophic factors [15]. The distance of neurite extension was 1.96 mm in 3 days (Fig. 8 1D). In addition, after postimplant in a rat T9 dorsal hemisection SCI model for 4 weeks, the aligned fibrin hydrogel was able to induce endogenous neural cell invasion and promote axonal outgrowth along the rapidly constructed aligned tissue cables in the lesion (Fig. 8 1E). As a result, the motor functional recovery was greatly promoted. Furthermore, the nanofiber hydrogel was applied to a large animal model by implanting the scaffold in a canine hemisected SCI model [16]. From hematoxylin and eosin staining, the growth of regenerative fascicular nerve fibers was observed in the aligned nanofiber hydrogel group (Fig. 8 1F), whereas the defect was still observed in the control group in which saline was filled in the lesion (Fig. 8 1G). From the immunofluorescence staining, the aligned fibrin nanofiber hydrogel also showed improvement on the regeneration of axons in comparison with the control group. By further incorporating with growth factors and cells, this type of scaffold will be promising for the repair of SCI. Due to the electrically intrinsic nerve in the native tissue, electrical stimulation is another efficient strategy to further improve the nerve regeneration. With traditional conductive carbon materials, the long-term electrical stimulation on nerve growth and development can be limited. To address this issue, soft graphene nanofibers were fabricated by controlled assembly of graphene oxide sheets onto the surface of electrospun poly(vinyl chloride) nanofibers [17]. The ultrathin graphene sheath wrapped the entire surface of the individual nanofibers tightly. By culturing the rat spinal primary motor neurons on the graphene nanofibers under electrical stimulation of 100 mV/cm, unprecedented accelerated growth and development of the neurons were achieved. Self-assembling peptides characterized by their nanoscale architecture represent another type of promising nanomaterial to construct the nerve repair scaffold for axon regeneration with functional return of vision [18 20]. Self-assembling peptides can also be assembled with electrospun nanofibers into composite guidance channels for repairing the SCI. For example, the self-assembling peptide [RADA16-I-BMHP1 (Ac-RADARADARADARADAGGPFSSTKT-CONH2)] was assembled with the electrospun nanofibers made of a blend of poly(lactic-co-glycolic acid) (PLGA) and poly(ε-caprolactone) (PCL) to produce longitudinal guidance microchannels [21]. Fig. 8 2A and B shows the SEM images of the self-assembling peptides and the electrospun microguidance channel, respectively. After transplanting in a chronic SCI rat model (Fig. 8 2C), conspicuous cord reconstruction was observed 6 months later, and the nerve defect was replaced by newly formed tissue comprising neural and stromal cells over the whole length (c.2 mm) of the lesion (Fig. 8 2D), crossing both the rostral and the caudal channels/tissue interfaces.

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FIGURE 8–2 SEM images of (A) the self-assembled nanofibers and (B) an electrospun PCL/PLGA microguidance channel; a higher magnification is shown in the inset. (C) Schematic representation of the scaffold implantation. The conduit, filled with the self-assembling peptide via microsyringe injection (inset), was placed singularly within the cavity following scar ablation. An electrospun lamina was sutured and glued to the meninges. (D) Immunofluorescence staining of the neurofilament 200 positive fibers covered the whole length of the lesion, indicating the longitudinal reconstruction of transplanted cord crossing both the rostral and the caudal channels/ tissue interfaces (dotted line). PCL, poly(ε-caprolactone); PLGA, poly(lactic-co-glycolic acid); SEM, scanning electron microscopy. Reprinted with permission from F. Gelain, S. Panseri, S. Antonini, C. Cunha, M. Donega, J. Lowery, et al., Transplantation of nanostructured composite scaffolds results in the regeneration of chronically injured spinal cords, ACS Nano 5 (1) (2011) 227 236. © 2011, American Chemistry Society.

8.2.2 Nanomaterials Combined With Growth Factors for Repairing Spinal Cord Injury Neurotrophic factors play a crucial role in regulating the development and function of different sets of neurons of the CNS [10]. They are a family of polypeptide factors that support cell survival, promote cell growth and differentiation, and maintain normal cell function. To this end, neurotrophic factors are important for spinal cord recovery, neuronal survival and regeneration, synaptic regeneration, and limb motor function after SCI [22]. For example, by administering the following neurotrophic factors at the site of injury and transplantation: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4, ciliaryderived neurotrophic factor (CNTF), or vehicle alone, the capacity of mature CNS neurons for regrowth after injury was greatly enhanced [23]. Besides, some other bioactive agents

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have also shown effects on promoting nerve repair; for example, chondroitinase ABC (ChABC) could digest the neuronal growth-inhibitory glycosaminoglycan side chains of chondroitin sulfate proteoglycans [24,25]. Various types of bioactive factors have been incorporated in scaffolds to endow them with biologically active functionalities to promote nerve regeneration. The bioactive factors can be directly encapsulated in the nanofibers by electrospinning of the solution containing the growth factors or by coaxial electrospinning in which the solution containing the growth factors serves as the core fluid. The bioactive factors can also be immobilized on the surface of the electrospun nanofibers by physical adsorption or chemical crosslinking. In one study, aligned electrospun silk fibroin nanofibers were incorporated with CNTF and nerve growth factor (NGF) [26]. The CNTF significantly stimulated the growth and migration of the glial cells, and a threefold enhancement in the neurite length of DRG cultured on the nanofibers was observed. In another study, NT-3 and ChABC were coencapsulated in electrospun collagen nanofibers for repairing SCI [27]. The nanofibers with a sustained release of NT-3 could support the neuronal culture and neurites outgrowth for a longer period than the scaffold with bolus delivery of NT-3. The delivery of ChABC could digest the neuronal growth inhibitors and thus promoted neurite outgrowth. Other chemicals have also been incorporated with the scaffolds to further improve the repair efficiency. Except for neurotrophins, ibuprofen was also reported to be loaded in PCL nanofibers to enhance axonal regeneration in the context of a spinal cord lesion by limiting the inflammatory response, which is one of the most relevant targets in nerve regeneration strategies [28]. Retinoic acid and purmorphamine were also coloaded in the electrospun poly (l-lactide) (PLLA)/gelatin nanofibers for controlling the differentiation of neuronal stem cells into motor neurons [29]. The bioactive factors have also been incorporated in the hybrid scaffold made of nanofibers and hydrogel to combine the topographical cues, soft mechanical matrix, together with the biochemical cues for promoting nerve repair. In one recent study, a hybrid scaffold was fabricated by sparsely distributing 3D aligned poly(ε-caprolactone-co-ethyl ethylene phosphate) nanofibers within an NT-3-loaded collagen hydrogel [30]. After postimplantation in a C5 rat spinal cord incision injury for 3 months, the hybrid scaffold was able to guide neurite extension, leading to neovascularization formed into the lesion site in an organized and aligned manner.

8.2.3 Nanomaterials Combined With Cell Therapy for Repairing Spinal Cord Injury SCI is a very complex damage, involving different type of cells. In the neural tissue engineering therapy of SCI, the major goal is to achieve regrowth of axons, inhibition of apoptosis, and replacement of injured cells with oligodendrocytes to increase axon remyelination. A combination of cell therapy with the scaffold is needed at the appropriate time and on the correct target site to realize successful neural tissue engineering. The transplantation of cellular scaffold with new supporting cells can be an ideal strategy for replenishing the lost neurons and for myelin regeneration to connect the injured axons and stimulate them for

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regrowth to restore function [31]. Different types of cells, such as neural cells and stem cells [e.g., NSCs and mesenchymal stem cells (MSCs)], have been incorporated in the nanofibrous scaffolds to provide an ideal strategy for replenishing the lost neurons and restore function [11]. For example, MSCs can facilitate axonal regeneration, which is suitable for reducing and minimizing many pathophysiological consequences of SCI.

8.3 Nanomaterials for the Repair of Peripheral Nerve Injury PNI caused by trauma, burns, or surgical intervention is a very common clinical problem, which can result in permanent disability of motor function and sensory perception [32,33]. Compared with CNS, the PNS has a greater capacity for axonal regeneration after injury. However, the functional recovery of the nerve is still a great challenge, especially when it comes to the case of a large defect in thick nerves. For severe injuries with larger gaps, interposition of nerve grafts is required to bridge the gap and support axonal regeneration [34 36]. Autografts are the clinical “gold standard” for bridging peripheral nerve lesions. However, the limited supply of donor nerves makes it difficult to reconstruct complex nerve gaps. To this end, artificial nerve guidance conduits (NGCs), which are typically made of either natural or synthetic biomaterials, have been developed to bridge the gap between the proximal and distal stumps. The ends of the damaged nerve are inserted into the ends of a conduit that provides a suitable environment for axon regeneration. The conduit acts not only as a connecting bridge for the severed nerve ends but also as a protective sheath for the regenerating nerve [37]. In the concept of neural tissue engineering for repairing PNI, it is critical to develop tissue-engineered nerve conduits that consist of a neural scaffold with support cells and growth factors. In the PNS, Schwann cells produce a myelin sheath around nerve fibers and play a critical role in the regeneration of damaged peripheral nerve. As such, all the topographic and biochemical features of a native nerve can be included to maximize the proliferation of Schwann cells and the elongation of axons. The in vitro models are usually focused on various immortalized Schwann cell lines and neuron models such as PC12 cells and DRG isolated from chick embryo or rat. As the main glial cells of the PNS, Schwann cells can promote neural regeneration by secretion of neurotrophin factors and production of basement membrane that consists of ECM proteins. In the preclinical view, the study of PNI and regeneration also needs to be carried out in animal models due to the structural complexity of this organ which can be only partly simulated in vitro [38]. By far the most often used experimental model is based on injury of the sciatic nerve, in small animals (e.g., rat and rabbit) and large animals (e.g., sheep, pig, and dog).

8.3.1 Design of the Nerve Conduits for Repairing the Peripheral Nerve Injury During the PNI repair process, the growth cone at the tip of the regenerating axon extends into the surrounding environment to sense the surface topographic cues [39]. It is of

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importance for a nerve conduit to provide topographical cues for guiding the directional extension of the axon. Among the conduits made of various types of nanomaterials, electrospun nanofibers represent a promising class of nanomaterials to produce nanofibrous conduits that are capable of physiologically mimicking the native ECM of neural tissues, providing contact guidance of the neurite outgrowth, and improving the nerve injury repair [40]. The NGC can be simply constructed by rolling the electrospun nanofiber mat and then sealing the boundary with a polymer solution. Nanofibers and conduits have been optimized in terms of structure, composition, and architecture for achieving an effective repair of the nerve injury by promoting the Schwann cell growth and neurite extension both in vitro and in vivo. Regarding the structure, uniaxially aligned nanofibers have been widely utilized to provide contact guidance to cells and neurites. The aligned nanofibers can direct neurites to extend along the direction of fiber alignment with an enhancement of neurite length [41]. For example, when chicken DRG bodies were cultured on electrospun PCL nanofibers, the neurites preferentially outgrew along the uniaxially aligned nanofibers while they were evenly distributed on random nanofibers. The average lengths of the neurites extended from DRG cultured on aligned nanofibers and random nanofibers were about 1100 and 800 μm, receptively [42]. Interestingly, the neurites could also be directed to grow in a direction perpendicular to the fiber alignment [43]. In this case the direction of the neurites was affected by the interaction between the neurites and the fibers, which was determined by the fiber density, surface chemistry of the fibers, and the surface chemistry of the supporting substrate to the fibers. When DRG were cultured on free-standing nanofibers, by increasing the fiber density through increasing the nanofiber collection durations, the extension of neurites switched from parallel (Fig. 8 3A) to perpendicular (Fig. 8 3B) outgrowth. When bare glass coverslips or polyethylene glycol-coated glass coverslips were used as the underlying substrate of aligned nanofibers, as shown in Fig. 8 3C and D, respectively, the neurites extended perpendicularly to the direction of fiber alignment. However, after coating the high-density free-standing nanofibers with laminin, the extension of neurites turned to parallel to the fiber alignment (Fig. 8 3E). A similar effect was also observed after surface coating with laminin on the substrate and the nanofibers, as shown in Fig. 8 3F, the neurites extended along the fiber alignment. A strong interaction between neurites and nanofibers resulted in parallel growth along the fibers, while a poor interaction led to perpendicular growth. In addition, with the increase in laminin amount coated on the surface of nanofibers, the length of the neurites increased. Therefore the surface modification of synthetic PCL nanofibers is essential to improve neurite outgrowth along the fiber alignment. In another study, it was demonstrated that the covalently attached laminin on electrospun PCL nanofiber surface promoted neurite outgrowth from PC12 cells, whereas fiber scaffolds containing higher protein concentrations contributed to significantly longer neurites [44]. Peptides were also noncovalently bonded to the electrospun PCL conduit through self-assembly, enhancing neurite outgrowth from DRG and promoting sciatic nerve regeneration [45]. The diameter of the nanofibers is also a significant factor that affects nerve regeneration [46]. In one study, the nanofibrous conduits comprised of uniaxially aligned electrospun PCL

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FIGURE 8–3 Fluorescence micrographs showing the neurite fields of DRG cultured on aligned nanofibers under different conditions. (A and B) DRG cultured on free-standing scaffolds of uniaxially aligned PCL nanofibers that were prepared by collecting for (A) 4 min and (B) 15 min, respectively. (C and D) DRG cultured on the nanofibers collecting for 15 min on (C) bare glass coverslips and (D) PEG precoated glass coverslips. (E and F) DRG cultured on nanofibers with laminin coating on (E) the surface of the free-standing nanofibers and (F) both the nanofibers and the underlying glass coverslips. The arrow in (A) implies the direction of the alignment for the underlying nanofibers in (A F). All samples were stained with antineurofilament 200. DRG, Dorsal root ganglia; PCL, poly (ε-caprolactone); PEG, polyethylene glycol. Reprinted with permission from J. Xie, W. Liu, M.R. MacEwan, P.C. Bridgman, Y. Xia, Neurite outgrowth on electrospun nanofibers with uniaxial alignment: the effects of fiber density, surface coating, and supporting substrate, ACS Nano 8 (2) (2014) 1878 1885. © 2014, American Chemistry Society.

microfibers (981 6 83 nm in diameter) or nanofibers (251 6 32 nm in diameter) were fabricated [46]. After implant into a 15-mm critical defect gap in a rat sciatic nerve injury model, nanofiber conduits resulted in a significantly higher total number of myelinated axons and thicker myelin sheaths than the microfiber conduits, indicating enhanced nerve regeneration and functional recovery. The material composition of the nanofibers also affects the neurite outgrowth from the nanofibers. Different natural materials (e.g., laminin, collagen, gelatin, and silk) can be combined with synthetic material to provide biochemically relevant signals for facilitating the regeneration speed and functional recovery. As one of the major components of the ECM in natural nerve, laminin provides an important pathfinding cue for the growth cones of developing neurons. For example, laminin was blended with PCL to fabricate electrospun nanofibers mimicking the peripheral nerve basement membrane [47]. In comparison with pure PCL nanofibers, by incorporating 10 wt.% laminin in the blend nanofibers, a significantly higher amount of PC12 cells was attached to the nanofibers, and longer neurite outgrowth

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was achieved for the DRG bodies on the nanofibers [47]. The mat comprised of aligned laminin PCL nanofibers was then inserted into the lumen of PCL microfiber conduit to conduct a rat tibial nerve transection recovery. At 6 weeks postimplantation, the presence of nanofibers within the lumen of the conduit promoted the nerve outgrowth and functional recovery in terms of nerve conduction. The enhancement could be attributed to the similarity of both the composition (laminin content) and the structure (alignment) of the native healing environment provided by this construct. As the major natural polymers in native peripheral nerves, collagen I and collagen III have also been explored to improve the nerve regeneration. By incorporating collagen with electrospun blend nanofibers of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), the nanofibers could promote the proliferation as well as neurotrophin secretion and glial cell line-derived neurotrophic factor (GDNF) gene expression of Schwann cells, leading to the regeneration of the myelin sheath [48]. By blending gelatin with electrospun poly(glycerol sebacate) nanofibers, the proliferation of PC12 cells was improved [49]. The incorporation of silk fibroin into aligned electrospun P(LLA-CL) nanofibers also supported greater Schwann cell proliferation [50]. It is worth pointing out that the influence of the materials composition on in vivo nerve repair might be different from the corresponding in vitro result. The mechanical properties and the degradation kinetics of the conduit made of electrospun nanofibers also need to be comprehensively considered. From one research [51], even though the in vitro studies demonstrated that the inclusion of gelatin into random electrospun nanofibers positively influenced the response of neurite extension from DRG, the in vivo studies in a rat sciatic nerve defect model showed an opposite response. The PCL conduits stimulated superior nerve regeneration than PCL/gelatin conduits as confirmed by electrophysiology, muscle weight, and histology, which was ascribed to the limited mechanical performance and degradation kinetics of the PCL/gelatin conduits. Even though different combinations of the material compositions in the electrospun nanofibers have been investigated as nerve repair scaffolds, the optimized composition cannot be concluded based on the present studies because of the lack of consolidated animal repair models and systematical comparison. Mimicking bioelectrical features of the peripheral nerve is another current strategy in neural tissue engineering that shows promising results. Studies have demonstrated that short-term, low-magnitude electrical stimulation correlates with enhanced growth and regeneration after PNI [52 54]. Much attention has been paid in designing electrical active scaffolds for neural regeneration. Different materials have been incorporated to endow the nanofibrous conduit with electrical property, such as by incorporation of conductive polymers [e.g., polyaniline (PANi), polypyrrole (PPy), and poly(3,4-ethylenedioxythiophene)] and carbon materials (e.g., carbon nanotubes, graphene oxide, and graphene). Under electrical stimulation, the nanofiber scaffold that blended with PANi [55] or surface coated with PPy [56] could induce neurite extension from PC12 cells without NGF treatment. In one study, PPy was coated on aligned PLLA nanofibers by in situ chemical oxidation polymerization, resulting in a conductive nanofiber mat with a resistivity of 0.874 Ω m at the direction of fiber alignment [57]. Under an electrical stimulation of 200 mV/cm, the median neurite lengths of PC12 differentiated on the aligned fibers were increased to 149.39 μm (Fig. 8 4A and C) in

FIGURE 8–4 Fluorescent images of PC12 cells cultured on aligned PPy-PLLA nanofibers without (A, C) and with (B, D) an electrical stimulation of 200 mV/cm. (E) Schematic of axon elongation from PC12 cells on aligned fibers after differentiation which involved the change of growth cone, and their inner change of filopodia during the elongation. (F) The in vivo performance of nanofiber conduits containing PPy after implanted in 10 mm rat sciatic nerve defect: representative images of toluidine blue staining in sections (1-μm thick) and the transmission electron microscopy images of the regenerative nerve at 3-month post-surgery. PLLA, Poly(l-lactide); PPy, polypyrrole. (A E) Reproduced with permission from Y. Zou, J. Qin, Z. Huang, G. Yin, X. Pu, D. He, Fabrication of aligned conducting PPy-PLLA fiber films and their electrically controlled guidance and orientation for neurites, ACS Appl. Mater. Interfaces 8 (20) (2016) 12576 12582. © 2016, American Chemical Society. (F) Reproduced with permission from Z.-F. Zhou, F. Zhang, J.-G. Wang, Q.-C. Chen, W.-Z. Yang, N. He, et al., Electrospinning of pela/ppy fibrous conduits: promoting peripheral nerve regeneration in rats by self-originated electrical stimulation, ACS Biomater. Sci. Eng. 2 (9) (2016) 1572 1581. © 2016, American Chemical Society.

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comparison with 114.73 μm in the case of the nonstimulated group (Fig. 8 4B and D). The mechanism of neurite extension on the aligned fibers under electrical stimulation was proposed, as schematized in Fig. 8 4E. The exerted electrical stimulation resulted in the enrichment of electric charges on the tops of the PPy particles and thus improved the aggregation of adhesion receptors and the interaction of adhesion receptors at the leading edge of filopodia with adhesion molecules. With the energy provided by the aggregated chondriosome in the microtubule growth channel, the assemble actin pulled the microtubules for elongation and filopodia extension to form a new cytoplasmic domain of the axon. From another in vivo study, nerve conduits made of poly(ethylene glycol)-co-poly(D,L-lactide) (PELA) and PPy (20%, 30%, and 50%) (PELA 2 PPy) were implanted for regeneration of a 10-mm rat sciatic nerve defect [58]. From the gross morphology and the transmission electron microscopy images of the regenerated nerves after 12 weeks (Fig. 8 4F), the structures of myelinated fibers in the PELA 2 PPy conduits were very similar to those in the autograft group; both groups showed much better performance than the group with plain PELA group. Owing to the unique electrical property, carbon nanomaterials are currently considered as another important series of nanomaterials for nerve regeneration [59,60]. Electrospun nanofibrous conduits made of nonconductive biopolymers have been hybridized with graphene, leading to the enhanced neurite outgrowth from PC12 cells [61,62]. Although the electrical stimulation results are encouraging, the stimulation process is often complicated. In most cases, the electrical stimulation is an invasive process, during which two electrodes are inserted to near the proximal and distal ends of the implanted guidance conduit, respectively, to serve as the positive and negative stimulating sites [63,64]. The depth of insertion varied from 1 to several centimeters according to the thickness of skin and fatty tissues [64]. In addition, the stimulation is often required to be applied several times in a designed interval to achieve the desired treatment outcome. To this end, the complicated delivery method of stimulation remains a challenge, especially in translating this technology for clinical application. Recently, conductive and electrically active scaffolds have been tested in vivo for functional peripheral nerve regeneration. In this approach, there is no need to implant a device to apply electrical stimulation, and the electroconductive scaffold itself can mimic the bioelectrical properties of the native tissue. Still, this approach needs to be combined with biomimetic chemical and topographical properties because electrical stimulation is most effective when delivered synergistically with other chemical biological factors. In addition to fabricating the conduit with a “roll & seal” method, other strategies have been developed to improve the reproducibility of the conduit and to further improve the nerve repair effect. Recently, a nanofiber conduit has also been prepared by directly electrospinning polymer solution onto two uniaxially aligned and horizontally oriented electrodes to produce a seamless conduit [65]. In addition, a single tubular conduit has limitations in supporting the nerve growth through the entire lumen, especially at the center part of the hollow lumen. Therefore a single tubular conduit is usually used to repair the nerve with a small diameter in a short defect. For the regeneration of a long defect in thick nerves, the single tubular conduit showed insufficient axon regeneration and limited functional recovery

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due to the poor intraluminal microenvironments. In this case, multichannel nerve guide conduit that can mimic the fascicular architecture of the native nerve proved to be very helpful [66]. For example, a conduit with multiple channels could be fabricated using a templated method [66]. The channel diameters, the wall thicknesses, and the number of the channels could be easily tailored. In vitro culture with Schwann cells revealed the longitudinal and transverse distribution of cell nuclei in the multichannel nerve guide. In another study, nanofiber-based multitubular conduits with a honeycomb structure were developed [67]. A bilayer mat of electrospun nanofibers was rolled up to form a single tube, with the inner and outer layers comprised of aligned and random nanofibers, respectively. Seven such tubes were then assembled into a hexagonal array and encased within the lumen of a larger tube to form the multitubular conduit (Fig. 8 5A C). The diameter of the conduit could reach around 5.0 mm, while the length could be easily tailored by changing the size of the mat utilized to form the tube. The seeded bone marrow stem cells (BMSCs) were able to proliferate in all the tubes with even circumferential and longitudinal distributions (Fig. 8 5D and E). Under chemical induction, the BMSCs were transdifferentiated into Schwann-like cells in all the tubes. The multiple parallel channels provide a greater number of defined paths and increased surface area compared to cylindrical guides.

FIGURE 8–5 (A C) SEM images of the multitubular conduit showing that seven small tubes were assembled into a hexagonal array and encased within the lumen of a larger tube. (D and E) Fluorescence micrographs showing that the BMSCs were able to proliferate in all the tubes with even (D) circumferential and (E) longitudinal distributions distributed in the multitubular conduit with a length of 4.2 cm after incubation for 7 days. BMSC, Bone marrow stem cell; SEM, scanning electron microscopy. Reproduced with permission from J. Xue, H. Li, Y. Xia, Nanofiberbased multi-tubular conduits with a honeycomb structure for potential application in peripheral nerve repair, Macromol. Biosci. 18 (9) (2018) 1800090. © 2018, John Wiley and Sons.

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The lumen of the conduit can also be included with nanomaterial-based fillers, in the form of aligned nanofibers [68], sponges, or gels, to provide an artificial microenvironment for guiding axonal regeneration. For example, aligned nanofiber yarns were assembled as the filler in a conduit comprised of random nanofibers [69]. The Schwann cells cultured in the conduit were distributed throughout the entire lumen. By incorporating laminin into the yarns, the proliferation of Schwann cells was further improved, indicating the synergistic effect of the topological structure and biological cues [70]. In addition, gel fillers such as selfassembling peptide hydrogels have been shown to be beneficial for improving regeneration by mimicking the high-water content and low stiffness of native ECM [71]. For example, the hierarchically aligned fibrin nanofiber hydrogel mentioned earlier has also been investigated for repair of injury to peripheral nerves [33]. The “nanofiber hydrogel” could induce the directional adhesion and migration of Schwann cells. After utilizing the nanofiber hydrogel as the intraluminal substrate in a bioengineered chitosan tube, the as-obtained conduit was implanted to bridge a 10-mm-long sciatic nerve gap in rat. Within 2 weeks, the aligned nanofiber hydrogel served as a beneficial microenvironment to support the cable formation of Schwann cells and axonal regrowth. After 12 weeks, the histological analyses showed that the regenerated axons in the group with the nanofiber hydrogel reached the distal stump. The density of regenerated nerve fibers at the distal end with the use of the aligned nanofiber hydrogel is compatible with the group with autograft. From the middle of the implants, with the incorporation of the nanofiber hydrogel, the myelinated nerve fiber density, the mean diameter of myelinated nerve fibers, and the thickness of the myelin sheath were all significantly higher in the group of the conduit filled with the nanofiber hydrogel in comparison with the hollow conduit.

8.3.2 Nanomaterials Combined With Growth Factors for Repairing Peripheral Nerve Injury For the repair of PNI, the provision of a sufficient amount of suitable growth factors is also critically important to facilitate and stimulate cell growth and axonal elongation. For example, by encapsulating NGF in the core of the PLLA-silk fibroin core sheath nanofibers, the PC12 cells could be simulated to extend neurites [69]. In another study, NGF was encapsulated in uniaxially aligned nanofibers made of a blend of silk fibroin and P(LLA-CL). From an in vivo rat sciatic nerve injury model, the group with the NGF greatly promoted regeneration of the nerve in comparison with the group without the NGF [72]. Different types of growth factors, such as BDNF and vascular endothelial growth factor (VEGF), can be coencapsulated in the scaffold [73]. The order of the released growth factor showed an influence on cell growth and nerve regeneration [74]. In one study, two types of silk fibroin-based scaffolds with controlled delivery order of VEGF and BDNF were prepared by placing one of the factors in the core while the other was in the sheath using coaxial electrospinning [74]. From an in vitro result, the inner-VEGF/outer-BDNF scaffolds could accelerate Schwann cell growth, proliferation, and spreading owing to the rapid release of BDNF. However, the in vivo result demonstrated that the inner-BDNF/outer-VEGF scaffolds significantly

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facilitated the angiogenesis and promoted the nerve regeneration. Furthermore, the growth factors could be combined with electrical stimulation to provide a synergistic effect for promoting neurite extension. In one study, NGF was encapsulated in the core of core sheath nanofibers. The core was made of a blend of poly(L-lactic acid-co-3-caprolactone) and silk fibroin, and the sheath was made of PANi. Upon the effect of electrical stimulation and controlled release of NGF, the outgrowth of neurites from PC12 cells cultured on the nanofibers was significantly enhanced compared with those on the plain nanofibers [56]. In the developing nervous system, pathfinding by growing axons may be guided by gradients of extracellular guidance factors [75]. Gradients of growth factors on the surface of scaffolds have been shown to be capable of enhancing and guiding neurite extension. In one study, a dual gradient of NGF and fiber density was developed along silk nanofibers [76]. On the nanofibers with uniform NGF concentration, the neurites extended from the neuron aligned in the two directions provided by the underlying nanofibers. In the gradient conditions, the neurites preferred to extend along the direction with the increase of NGF concentration. Moreover, on the nanofibers with NGF content gradient, the average neurite length was 417.6 6 55.7 μm, which was much higher than that of 264.5 6 37.6 μm on the nanofibers with uniform NGF content. Nanoparticles were also reported as one of the main vehicles to deliver growth factors. For example, magnetic composite nanoparticles were developed by combining NGF-loaded PLLA nanoparticles with magnetic iron oxide nanoparticles [77]. The PLLA fibers could direct the outgrowth of extending neurites DRG, aligning them in the direction of fiber orientation. After combining the composite nanoparticles with aligned PLLA microfibers by placing the composite particles 5 mm from the DRG body, the neurites extended toward the NGF gradient and grew farther (1703 6 125 μm) than those extended away from the direction of the nanoparticles (1353 6 90 μm). These studies demonstrated that chemotropic gradients and aligned topography are potent mechanisms that can be utilized to direct extending neurites.

8.3.3 Nanomaterials Combined With Cell Therapy for Repairing Peripheral Nerve Injury As one of the major cells in the native peripheral nerve, Schwann cells can be introduced prior to nerve regeneration so they are able to promote neurite growth by secreting neurotrophic molecules. A combination of scaffold and Schwann cells has shown a marked improvement in nerve regeneration and function recovery. For example, a tissue-engineered NGC based on double-layered electrospun nanofibers (aligned nanofiber in the top layer and random nanofibers in the bottom layer) was seeded with Schwann cells [78]. Fig. 8 6A and B shows fluorescence micrograph of the typical neurite field extending from DRG seeded on the double-layered PCL nanofibers in the absence and presence of preseeded Schwann cells, respectively. In the absence of Schwann cells, only some short neurites were projected from the cell body. However, a significantly larger number of neurites was observed from the group where Schwann cells were preseeded on the nanofibers. The average lengths of the neurites for the DRG cultured in the absence and presence of preseeded Schwann cells were 144 6 46 and 1843 6 137 μm, respectively, demonstrating that Schwann cells enhanced the

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FIGURE 8–6 Fluorescence micrographs of the typical neurite field extending from DRG seeded on double-layered PCL nanofiber scaffolds with the (A) absence and (B) presence of preseeded Schwann cells. The neurites were stained with Tuj1 marker (green). (C) Double staining of the Schwann cells in (B) with anti-S100 (red) and their nuclei with DAPI (blue). (D) Triple staining of the Schwann cells with anti-S100 (red), their nuclei with DAPI (blue), and the neurites with Tuj1 marker (green) for the sample shown in (B). (E) Schematic of the bilayer NGCs used for the microsurgical repair of a critical nerve, and the representative histological sections of the regenerated peripheral nerve tissue harvested 5 mm distal to interposed (F) fresh nerve isograft, (G) bilayer NGC, and (H) bilayer NGC preseeded with Schwann cells at 12 weeks postoperation. DRG, Dorsal root ganglia; DAPI, 4',6-diamidino-2phenylindole; NGC, nerve guidance conduit. Reproduced with permission from J. Xie, M.R. MacEwan, W. Liu, N. Jesuraj, X. Li, D. Hunter, Y. Xia, Nerve guidance conduits based on double-layered scaffolds of electrospun nanofibers for repairing the peripheral nervous system, ACS Appl. Mater. Interfaces 6 (12) (2014) 9472 9480. © 2014, American Chemical Society.

outgrowth of neurites. From the double staining of the Schwann cells in Fig. 8 6C and the tristaining of the Schwann cells as well as the neurites from DRG in Fig. 8 6D, the neurites were preferentially extended along both the elongated Schwann cells and aligned nanofibers. The efficiency of the bilayer NGCs in facilitating nerve regeneration was evaluated through the microsurgical repair of a critical nerve gap of 14 mm for 12 weeks. Three experimental groups were examined, including isografts and bilayer NGCs with and without preseeded Schwann cells. Fig. 8 6E H shows the histomorphometric analysis of the regenerated peripheral nerve tissue 12 weeks postoperation using different types of conduits. While the isograft group outperformed both NGC groups, the presence of Schwann cells inside bilayer NGCs could greatly promote the sprouting of regenerating nerve fibers. By changing the material composition of the electrospun fibers, NGC composed of a polymer gold nanoparticle nanocomposite was preseeded with Schwann cells to repair the peripheral nerve [79]. The in vivo function of the conduit was tested in a neurotmesis grade sciatic nerve injury model in rat over a period of 18 months. Compared with the plain conduit, in the conduit preseeded with Schwann cells, a large amount of Schwann cells were recruited inside the lumen as well as within the interlayer gaps of the conduit and aligned in characteristic wave-like fashion. The near normal values of nerve conduction velocity (50 m/s), compound muscle action potential (29.7 mV), and motor unit potential (133 mV) of the nerves repaired by the cellular conduits were superior to those observed in previous reports with synthetic materials as well as collagen-based nerve conduits.

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The harvest of Schwann cells limits its application as a cellular resource in clinical applications, so combining stem cell therapy with an electrospun fibrous conduit would be the most promising tissue-engineered NGC [52,80]. Various types of stem cells have been studied as cellular resources for tissue-engineered NGC. In one study, stem cells from human exfoliated deciduous tooth were transplanted to the site of sciatic nerve injury through the NGC made of electrospun PCL/gelatin nanofibers [51]. For comparison, nerve regeneration in sutured sciatic nerve and untreated injured sciatic nerve was also examined. While both NGCs supported axonal regeneration across the nerve gap, regeneration through NGC with the cells was found to be superior in terms of nerve regrowth, functional and sensory recovery, and histological assessment. The stem cells, including embryonic stem cells, NSCs, adipose-derived stem cells, ectomesenchymal stem cells, and BMSCs, can also be transdifferentiated into Schwann cells on the nanofibers, proving an alternative source of the limited Schwann cells. For example, by optimizing the alignment, diameter, and surface property, electrospun PCL nanofibers could support the differentiation of BMSCs into Schwann cells under the stimulation of growth factor cocktails [78]. The cellular nanofibers could stimulate neurite extension from both the PC12 cells and the DRG bodies.

8.4 Conclusions This chapter presents a recent progress on the design and test of nanomaterials for neural tissue engineering by focusing on the repair of SCI and PNI. Nanomaterials are currently being utilized for tissue engineering, due to their capability of closely mimicking the tissuespecific bioenvironments in terms of the composition and structure of ECM of the native tissue. Nanofibrous nerve grafts can be constructed by electrospinning and self-assembly peptides. The physical parameters, composition, mechanical strength, and surface chemistry can be well tailored for promoting cell growth and axon outgrowth. Nanofibrous scaffolds have also been combined with nanoparticles to manipulate the cell migration, proliferation, and differentiation and guide neurite extension, aiming to facilitate the axonal elongation and functional recovery. The growth factors and cell therapy are needed to be combined with the nanomaterial scaffold to enhance the outcome of tissue repair. Tissue-engineered nanomaterials represent the new generation of implant for the repair of nerve injury. Even though scaffold made from nanomaterials offers impressive development for neural tissue regeneration, challenges still exist. One challenge is the difficulty of producing uniform nanofibers with diameters below 50 nm, which is desirable to better mimic the ECM. Another challenge is the construction of 3D nanofibrous scaffold with ordered structure and controlled pore size to regulate cell infiltration and thus the integration of the repaired neural tissue with the native tissue. In addition, it is necessary to appropriately combine the neurotrophic factors and cell therapy with the structural design of the scaffolding material in a complementary manner to improve the nerve repair. These challenges will provide new opportunities to design and optimize the scaffolding nanomaterials to push them toward clinical applications in neural tissue engineering.

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Acknowledgments This work was supported by a grant from the National Institutes of Health (R01 EB020050).

References [1] B. Kolb, I.Q. Whishaw, Fundamentals of Human Neuropsychology, sixth ed., Macmillan, New York, 2009. [2] J. Isaksson, P. Kjäll, D. Nilsson, N. Robinson, M. Berggren, A. Richter-Dahlfors, Electronic control of Ca21 signalling in neuronal cells using an organic electronic ion pump, Nat. Mater 6 (9) (2007) 673 679. [3] C.E. Schmidt, J.B. Leach, Neural tissue engineering: strategies for repair and regeneration, Annu. Rev. Biomed. Eng. 5 (1) (2003) 293 347. [4] L. Zhang, T.J. Webster, Nanotechnology and nanomaterials: promises for improved tissue regeneration, Nano Today 4 (1) (2009) 66 80. [5] G.A.A. Saracino, D. Cigognini, D. Silva, A. Caprini, F. Gelain, Nanomaterials design and tests for neural tissue engineering, Chem. Soc. Rev. 42 (1) (2013) 225 262. [6] J. Wu, L. Xie, W.Z.Y. Lin, Q. Chen, Biomimetic nanofibrous scaffolds for neural tissue engineering and drug development, Drug Discov. Today 22 (9) (2017) 1375 1384. [7] S. Mobini, B.S. Spearman, C.S. Lacko, C.E. Schmidt, Recent advances in strategies for peripheral nerve tissue engineering, Curr. Opin. Biomed. Eng. 4 (2017) 134 142. [8] S.A. Busch, J. Silver, The role of extracellular matrix in CNS regeneration, Curr. Opin. Neurobiol. 17 (1) (2007) 120 127. [9] K. Pawar, B.J. Cummings, A. Thomas, L.D. Shea, A. Levine, S. Pfaff, et al., Biomaterial bridges enable regeneration and re-entry of corticospinal tract axons into the caudal spinal cord after SCI: association with recovery of forelimb function, Biomaterials 65 (2015) 1 12. [10] H.M. Tuinstra, M.O. Aviles, S. Shin, S.J. Holland, M.L. Zelivyanskaya, A.G. Fast, et al., Multifunctional, multichannel bridges that deliver neurotrophin encoding lentivirus for regeneration following spinal cord injury, Biomaterials 33 (5) (2012) 1618 1626. [11] C. Fan, X. Li, Z. Xiao, Y. Zhao, H. Liang, B. Wang, et al., A modified collagen scaffold facilitates endogenous neurogenesis for acute spinal cord injury repair, Acta Biomater. 51 (2017) 304 316. [12] K. Pawar, R. Mueller, M. Caioni, P. Prang, U. Bogdahn, W. Kunz, et al., Increasing capillary diameter and the incorporation of gelatin enhance axon outgrowth in alginate-based anisotropic hydrogels, Acta Biomater. 7 (7) (2011) 2826 2834. ´ [13] E. Kijenska, M.P. Prabhakaran, W. Swieszkowski, K.J. Kurzydlowski, S. Ramakrishna, Electrospun biocomposite P(LLA-CL)/collagen I/collagen III scaffolds for nerve tissue engineering, J. Biomed. Mater. Res. Part B 100 (4) (2012) 1093 1102. [14] T. Liu, J.D. Houle, J. Xu, B.P. Chan, S.Y. Chew, Nanofibrous collagen nerve conduits for spinal cord repair, Tissue Eng. Part A 18 (9 10) (2012) 1057 1066. [15] S. Yao, X. Liu, S. Yu, X. Wang, S. Zhang, Q. Wu, et al., Co-effects of matrix low elasticity and aligned topography on stem cell neurogenic differentiation and rapid neurite outgrowth, Nanoscale 8 (19) (2016) 10252 10265. [16] Z. Zhang, S. Yao, S. Xie, X. Wang, F. Chang, J. Luo, et al., Effect of hierarchically aligned fibrin hydrogel in regeneration of spinal cord injury demonstrated by tractography: a pilot study, Sci. Rep. 7 (2017) 40017.

206

THERANOSTIC BIONANOMATERIALS

[17] Z.Q. Feng, T. Wang, B. Zhao, J. Li, L. Jin, Soft graphene nanofibers designed for the acceleration of nerve growth and development, Adv. Mater. 27 (41) (2015) 6462 6468. [18] F. Gelain, D. Bottai, A. Vescovi, S. Zhang, Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures, PLoS One 1 (1) (2006) e119. [19] R.G. Ellis-Behnke, Y.-X. Liang, S.-W. You, D.K.C. Tay, S. Zhang, K.-F. So, et al., Nano neuro knitting: peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision, Proc. Natl. Acad. Sci. USA 103 (13) (2006) 5054 5059. [20] Y. Sun, W. Li, X. Wu, N. Zhang, Y. Zhang, S. Ouyang, et al., Functional self-assembling peptide nanofiber hydrogels designed for nerve degeneration, ACS Appl. Mater. Interfaces 8 (3) (2016) 2348 2359. [21] F. Gelain, S. Panseri, S. Antonini, C. Cunha, M. Donega, J. Lowery, et al., Transplantation of nanostructured composite scaffolds results in the regeneration of chronically injured spinal cords, ACS Nano 5 (1) (2011) 227 236. [22] H. Fang, C. Liu, M. Yang, H. Li, F. Zhang, W. Zhang, et al., Neurotrophic factor and Trk signaling mechanisms underlying the promotion of motor recovery after acute spinal cord injury in rats, Exp. Ther. Med. 14 (1) (2017) 652 656. [23] B.S. Bregman, M. McAtee, H.N. Dai, P.L. Kuhn, Neurotrophic factors increase axonal growth after spinal cord injury and transplantation in the adult rat, Exp. Neurol. 148 (2) (1997) 475 494. [24] E.J. Bradbury, L.D. Moon, R.J. Popat, V.R. King, G.S. Bennett, P.N. Patel, et al., ABC promotes functional recovery after spinal cord injury, Nature 416 (6881) (2002) 636. [25] J.R. Siebert, A. Conta Steencken, D.J. Osterhout, Chondroitin sulfate proteoglycans in the nervous system: inhibitors to repair, BioMed Res. Int. 2014 (2014) 15. ID 845323. [26] T.M. Dinis, G. Vidal, R.R. Jose, P. Vigneron, D. Bresson, V. Fitzpatrick, et al., Complementary effects of two growth factors in multifunctionalized silk nanofibers for nerve reconstruction, PLoS One 9 (10) (2014) e109770. [27] T. Liu, J. Xu, B.P. Chan, S.Y. Chew, Sustained release of neurotrophin-3 and Chondroitinase ABC from electrospun collagen nanofiber scaffold for spinal cord injury repair, J. Biomed. Mater. Res. Part A 100 (1) (2012) 236 242. [28] L.R. Pires, V. Guarino, M.J. Oliveira, C.C. Ribeiro, M.A. Barbosa, L. Ambrosio, et al., Ibuprofen-loaded poly (trimethylene carbonate-co-ε-caprolactone) electrospun fibres for nerve regeneration, J. Tissue Eng. Regen. Med. 10 (13) (2013) E154 E166. [29] L. Binan, C. Tendey, G. De Crescenzo, R. El Ayoubi, A. Ajji, M. Jolicoeur, Differentiation of neuronal stem cells into motor neurons using electrospun poly-L-lactic acid/gelatin scaffold, Biomaterials 35 (2) (2014) 664 674. [30] U. Milbreta, L.H. Nguyen, H. Diao, J. Lin, W. Wu, C.-Y. Sun, et al., Three-dimensional nanofiber hybrid scaffold directs and enhances axonal regeneration after spinal cord injury, ACS Biomater. Sci. Eng. 2 (8) (2016) 1319 1329. [31] A. Muheremu, J. Peng, Q. Ao, Stem cell based therapies for spinal cord injury, Tissue Cell 48 (4) (2016) 328 333. [32] V. Mukhatyar, L. Karumbaiah, J. Yeh, R. Bellamkonda, Tissue engineering strategies designed to realize the endogenous regenerative potential of peripheral nerves, Adv. Mater. 21 (46) (2009) 4670 4679. [33] J. Du, J. Liu, S. Yao, H. Mao, J. Peng, X. Sun, et al., Prompt peripheral nerve regeneration induced by a hierarchically aligned fibrin nanofiber hydrogel, Acta Biomater. 55 (2017) 296 309. [34] H. Cao, T. Liu, S.Y. Chew, The application of nanofibrous scaffolds in neural tissue engineering, Adv. Drug Del. Rev. 61 (12) (2009) 1055 1064. [35] S. Kehoe, X. Zhang, D. Boyd, FDA approved guidance conduits and wraps for peripheral nerve injury: a review of materials and efficacy, Injury 43 (5) (2012) 553 572.

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[36] X. Gu, F. Ding, D.F. Williams, Neural tissue engineering options for peripheral nerve regeneration, Biomaterials 35 (24) (2014) 6143 6156. [37] J. Xue, H. Li, Y. Xia, Nanofiber-based multi-tubular conduits with a honeycomb structure for potential application in peripheral nerve repair, Macromol. Biosci. 18 (9) (2018). 1800090. [38] S. Geuna, The sciatic nerve injury model in pre-clinical research, J. Neurosci. Methods 243 (2015) 39 46. [39] D. Hoffman-Kim, J.A. Mitchel, R.V. Bellamkonda, Topography, cell response, and nerve regeneration, Annu. Rev. Biomed. Eng. 12 (2010) 203 231. [40] J. Xie, M.R. MacEwan, A.G. Schwartz, Y. Xia, Electrospun nanofibers for neural tissue engineering, Nanoscale 2 (1) (2010) 35 44. [41] C. Simitzi, A. Ranella, E. Stratakis, Controlling the morphology and outgrowth of nerve and neuroglial cells: the effect of surface topography, Acta Biomater. 51 (2017) 21 52. [42] J. Xie, M.R. MacEwan, X. Li, S.E. Sakiyama-Elbert, Y. Xia, Neurite outgrowth on nanofiber scaffolds with different orders, structures, and surface properties, ACS Nano 3 (5) (2009) 1151 1159. [43] J. Xie, W. Liu, M.R. MacEwan, P.C. Bridgman, Y. Xia, Neurite outgrowth on electrospun nanofibers with uniaxial alignment: the effects of fiber density, surface coating, and supporting substrate, ACS Nano 8 (2) (2014) 1878 1885. [44] N.E. Zander, J.A. Orlicki, A.M. Rawlett, T.P. Beebe Jr, Quantification of protein incorporated into electrospun polycaprolactone tissue engineering scaffolds, ACS Appl. Mater. Interfaces 4 (4) (2012) 2074 2081. [45] L. Zhu, K. Wang, T. Ma, L. Huang, B. Xia, S. Zhu, et al., Noncovalent bonding of RGD and YIGSR to an electrospun poly (ε-caprolactone) conduit through peptide self-assembly to synergistically promote sciatic nerve regeneration in rats, Adv. Healthcare Mater. 6 (8) (2017) 1600860. [46] X. Jiang, R. Mi, A. Hoke, S.Y. Chew, Nanofibrous nerve conduit-enhanced peripheral nerve regeneration, J. Tissue Eng. Regen. Med. 8 (5) (2014) 377 385. [47] R.A. Neal, S.S. Tholpady, P.L. Foley, N. Swami, R.C. Ogle, E.A. Botchwey, Alignment and composition of laminin polycaprolactone nanofiber blends enhance peripheral nerve regeneration, J. Biomed. Mater. Res. Part A 100 (2) (2012) 406 423. [48] E. Masaeli, M. Morshed, M.H. Nasr-Esfahani, S. Sadri, J. Hilderink, A. van Apeldoorn, et al., Fabrication, characterization and cellular compatibility of poly (hydroxy alkanoate) composite nanofibrous scaffolds for nerve tissue engineering, PLoS One 8 (2) (2013) e57157. [49] J. Hu, D. Kai, H. Ye, L. Tian, X. Ding, S. Ramakrishna, et al., Electrospinning of poly (glycerol sebacate)based nanofibers for nerve tissue engineering, Mater. Sci. Eng. C 70 (2017) 1089 1094. [50] K. Zhang, W. Jinglei, C. Huang, X. Mo, Fabrication of silk fibroin/P(LLA-CL) aligned nanofibrous scaffolds for nerve tissue engineering, Macromol. Mater. Eng. 298 (5) (2013) 565 574. [51] V. Cirillo, B.A. Clements, V. Guarino, J. Bushman, J. Kohn, L. Ambrosio, A comparison of the performance of mono-and bi-component electrospun conduits in a rat sciatic model, Biomaterials 35 (32) (2014) 8970 8982. [52] X. Zhang, N. Xin, L. Tong, X.-J. Tong, Electrical stimulation enhances peripheral nerve regeneration after crush injury in rats, Mol. Med. Report 7 (5) (2013) 1523 1527. [53] T. Gordon, Electrical stimulation to enhance axon regeneration after peripheral nerve injuries in animal models and humans, Neurotherapeutics 13 (2) (2016) 295 310. [54] C.E. Schmidt, V.R. Shastri, J.P. Vacanti, R. Langer, Stimulation of neurite outgrowth using an electrically conducting polymer, Proc. Natl. Acad. Sci. USA 94 (17) (1997) 8948 8953. [55] B. Sun, T. Wu, J. Wang, D. Li, J. Wang, Q. Gao, et al., Polypyrrole-coated poly (L-lactic acid-co-ε-caprolactone)/silk fibroin nanofibrous membranes promoting neural cell proliferation and differentiation with electrical stimulation, J. Mater. Chem. B 4 (41) (2016) 6670 6679.

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[56] J. Zhang, K. Qiu, B. Sun, J. Fang, K. Zhang, E.-H. Hany, et al., The aligned core sheath nanofibers with electrical conductivity for neural tissue engineering, J. Mater. Chem. B 2 (45) (2014) 7945 7954. [57] Y. Zou, J. Qin, Z. Huang, G. Yin, X. Pu, D. He, Fabrication of aligned conducting PPy-PLLA fiber films and their electrically controlled guidance and orientation for neurites, ACS Appl. Mater. Interfaces 8 (20) (2016) 12576 12582. [58] Z.-F. Zhou, F. Zhang, J.-G. Wang, Q.-C. Chen, W.-Z. Yang, N. He, et al., Electrospinning of PELA/PPy fibrous conduits: promoting peripheral nerve regeneration in rats by self-originated electrical stimulation, ACS Biomater. Sci. Eng. 2 (9) (2016) 1572 1581. [59] A. Fraczek-Szczypta, Carbon nanomaterials for nerve tissue stimulation and regeneration, Mater. Sci. Eng. C 34 (2014) 35 49. [60] F. Mottaghitalab, M. Farokhi, A. Zaminy, M. Kokabi, M. Soleimani, F. Mirahmadi, et al., A biosynthetic nerve guide conduit based on silk/SWNT/fibronectin nanocomposite for peripheral nerve regeneration, PLoS One 8 (9) (2013) e74417. [61] K. Zhang, H. Zheng, S. Liang, C. Gao, Aligned PLLA nanofibrous scaffolds coated with graphene oxide for promoting neural cell growth, Acta Biomater. 37 (2016) 131 142. [62] N. Golafshan, M. Kharaziha, M. Fathi, Tough and conductive hybrid graphene-PVA: alginate fibrous scaffolds for engineering neural construct, Carbon 111 (2017) 752 763. [63] M.C. Lu, C.Y. Ho, S.F. Hsu, H.C. Lee, J.H. Lin, C.H. Yao, et al., Effects of electrical stimulation at different frequencies on regeneration of transected peripheral nerve, Neurorehabil Neural Repair 22 (4) (2008) 367 373. [64] C.H. Kao, J. Chen, Y.M. Hsu, D.T. Bau, C.H. Yao, Y.S. Chen, High-frequency electrical stimulation can be a complementary therapy to promote nerve regeneration in diabetic rats, PLoS One 8 (11) (2013) e79078. [65] C. Huang, Y. Ouyang, H. Niu, N. He, Q. Ke, X. Jin, et al., Nerve guidance conduits from aligned nanofibers: improvement of nerve regeneration through longitudinal nanogrooves on a fiber surface, ACS Appl. Mater. Interfaces 7 (13) (2015) 7189 7196. [66] E.M. Jeffries, Y. Wang, Biomimetic micropatterned multi-channel nerve guides by templated electrospinning, Biotechnol. Bioeng. 109 (6) (2012) 1571 1582. [67] J. Xue, H. Li, Y. Xia, Nanofiber-based multi-tubular conduits with a honeycomb structure for potential application in peripheral nerve repair, Macromol. Biosci. 18 (9) (2018) 1800090. [68] Y. Gu, J. Zhu, C. Xue, Z. Li, F. Ding, Y. Yang, et al., Chitosan/silk fibroin-based, Schwann cell-derived extracellular matrix-modified scaffolds for bridging rat sciatic nerve gaps, Biomaterials 35 (7) (2014) 2253 2263. [69] L. Tian, M.P. Prabhakaran, J. Hu, M. Chen, F. Besenbacher, S. Ramakrishna, Coaxial electrospun poly (lactic acid)/silk fibroin nanofibers incorporated with nerve growth factor support the differentiation of neuronal stem cells, RSC Adv. 5 (62) (2015) 49838 49848. [70] T. Wu, D. Li, Y. Wang, B. Sun, D. Li, Y. Morsi, et al., Laminin-coated nerve guidance conduits based on poly(L-lactide-co-glycolide) fibers and yarns for promoting Schwann cells proliferation and migration, J. Mater. Chem. B 5 (17) (2017) 3186 3194. [71] A. Li, A. Hokugo, A. Yalom, E.J. Berns, N. Stephanopoulos, M.T. McClendon, et al., A bioengineered peripheral nerve construct using aligned peptide amphiphile nanofibers, Biomaterials 35 (31) (2014) 8780 8790. [72] K. Zhang, C. Wang, C. Fan, X. Mo, Aligned SF/P(LLA-CL)-blended nanofibers encapsulating nerve growth factor for peripheral nerve regeneration, J. Biomed. Mater. Res. Part A 102 (8) (2014) 2680 2691. [73] Q. Liu, J. Huang, H. Shao, L. Song, Y. Zhang, Dual-factor loaded functional silk fibroin scaffolds for peripheral nerve regeneration with the aid of neovascularization, RSC Adv. 6 (9) (2016) 7683 7691.

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[74] Y. Zhang, J. Huang, L. Huang, Q. Liu, H. Shao, X. Hu, et al., Silk fibroin-based scaffolds with controlled delivery order of VEGF and BDNF for cavernous nerve regeneration, ACS Biomater. Sci. Eng. 2 (11) (2016) 2018 2025. [75] G.-L. Ming, S.T. Wong, J. Henley, X.-B. Yuan, H.-J. Song, N.C. Spitzer, et al., Adaptation in the chemotactic guidance of nerve growth cones, Nature 417 (6887) (2002) 411 418. [76] T.M. Dinis, R. Elia, G. Vidal, A. Auffret, D.L. Kaplan, C. Egles, Method to form a fiber/growth factor dual-gradient along electrospun silk for nerve regeneration, ACS Appl. Mater. Interfaces 6 (19) (2014) 16817 16826. [77] J.M. Zuidema, C. Provenza, T. Caliendo, S. Dutz, R.J. Gilbert, Magnetic NGF-releasing PLLA/iron oxide nanoparticles direct extending neurites and preferentially guide neurites along aligned electrospun microfibers, ACS Chem. Neurosci. 6 (11) (2015) 1781 1788. [78] J. Xie, M.R. MacEwan, W. Liu, N. Jesuraj, X. Li, D. Hunter, et al., Nerve guidance conduits based on double-layered scaffolds of electrospun nanofibers for repairing the peripheral nervous system, ACS Appl. Mater. Interfaces 6 (12) (2014) 9472 9480. [79] S. Das, M. Sharma, D. Saharia, K.K. Sarma, M.G. Sarma, B.B. Borthakur, et al., In vivo studies of silk based gold nano-composite conduits for functional peripheral nerve regeneration, Biomaterials 62 (2015) 66 75. [80] M. Uz, S.R. Das, S. Ding, D.S. Sakaguchi, J.C. Claussen, S.K. Mallapragada, Advances in controlling differentiation of adult stem cells for peripheral nerve regeneration, Adv. Healthc. Mater (2018) 1701046.

Further Reading J. Xue, J. Yang, D.M. O’Connor, C. Zhu, D. Huo, N.M. Boulis, et al., Differentiation of bone marrow stem cells into Schwann cells for the promotion of neurite outgrowth on electrospun fibers, ACS Appl. Mater. Interfaces 9 (14) (2017) 12299 12310.

9 Nanomaterials for Wound Healing: Scope and Advances Juan Du, Kenneth K.Y. Wong DEPART ME NT OF SURGERY, THE UNIVERSITY OF HO NG KO NG , H O N G K O N G S AR , C HI N A

9.1 Introduction In clinical practice, wounds are encountered daily. Improving wound healing is thus a major challenge faced by surgeons and is a hot point of research. The common therapies include adequate surgical debridement, effective antibiotic therapy, proper moist dressings, and correction of metabolic abnormalities. These are essential for wound repair. In addition, hyperbaric oxygen therapy, electric stimulation, and negative-pressure wound therapy are also valuable adjuncts for healing, especially to improve some chronic wound outcomes [1]. Many new methods looking into improved wound healing have been proposed in basic laboratory research and clinical medicine since the 1990s. Nanomedicine is one of the newest branches in science and has been attracting the attention of many researchers. Nanomedicine, as an important crosslink between nanoscience and biomedicine, is defined as the process of disease prevention, diagnosis, and therapeutics using bionanomaterials or nanotechnology [2]. Bionanomaterials are applied broadly in the therapy of cancer [3], gene therapy, vaccine- and drug-delivery [4], and regenerative medicine (Fig. 9 1). Nanomedicine has also opened a new gateway to wound healing and tissue regeneration. Indeed, many basic medical research data and clinical trials with exciting results have already been published. A systematic evaluation of the use of bionanomaterials in wound healing from scope to advancement will be summarized in this chapter. The aim is to sum up the current knowledge of the mechanism of bionanomaterials in promoting wound healing and introduce their applications in clinical therapy.

9.2 Brief Introduction to Bionanomaterials on Wound Healing The original concept of nano means the scale of one nanometer (nm), which is equal to one billionth of a meter (1029 m), as well as with the manipulation of single atoms and molecules. Thus, materials on a nanoscale are named nanomaterials or bionanomaterials if used in biomedicine. For their effects, the smaller the particle, the larger the surface area-to-volume ratio. Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00009-2 © 2019 Elsevier Inc. All rights reserved.

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FIGURE 9–1 The framework of nanomedicine and its scope of applications.

FIGURE 9–2 Comparison of nanoscale and micron scale levels.

The large surface areas of nanoparticles can offer many potential benefits in designing multifunctional nanosystems with the loading of surface functional ligands. The size of bionanomaterials is usually controlled at 1 100 nm, which is very important in terms of having biological properties and functions. This is because of the similarity in size as compared to DNA (approximately 2.5 nm in width) and protein molecules (1 20 nm in width) (Fig. 9 2). For therapeutic applications, the exact sizes and surface characteristics of bionanomaterials can be tailored or controlled according to requirements. In addition, new technological developments can overcome difficulties which could not be achieved by conventional large-scale manufacturing processes. Different shapes of bionanomaterials exist, including dendrimers, spheres, rods, cubes, wires, and multifacet materials. These can be fabricated into nanofibers, nanocapsules, nanotubes, nanogels, as well as polymeric bionanomaterials. The nanostructure of the surface coating controls properties such as charge, conductivity, roughness, porosity, surface moisture , which are all important factors in the interaction with living cells of an organism. Nanoparticles can be synthesized through an array of methods: spark discharging, electrochemical reduction, solution irradiating, cryochemical synthesis, etc. Indeed, the

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manufacture of bionanomaterials has contributed to the rapid development of nanotechnology, which is becoming the driving force behind a variety of and revolutionary innovation in the medical field. Underlying knowledge on nanostructure surfaces has also developed rapidly. An example of this is the optimization of the interaction between nanomaterials on the surface of prostheses (such as artificial joints), and body tissues, with the aim of producing biomaterials which have close integration inside the host body. On the other hand, research has focused on the reduction of potential side effects of nanomaterials, such as induction of inflammation or allergy reactions. In the field of wound healing and regeneration, bionanomaterials currently in use include silver nanoparticles (AgNPs), nanogold (nano-Au) [5], TiO2 nanoparticles [6,7], nano ZnO [8], nanotantalum [9], nano CuO [10], lipid nanoparticles [3], carbon nanotubes, SiO2 nanoparticles [11], and nanochitosan.

9.3 Characteristics of Wound Healing: Normal and Abnormal In the clinical setting, the types of wounds encountered and their classification are vastly different. Wounds can be divided according to the anatomy in the body, and also to the mechanism of injury, that is, abrasion wound, laceration wound, excision wound, etc. Overall, wound healing is a complex biological process to restore the integrity of tissue after injury. Normal wound repair is characterized by four overlapping phases. (1) Coagulation or hemostasis—after injury, most wounds will bleed. The formation of a clot then serves as a temporary shield protecting the denuded wound tissues and provides a provisional matrix over and through which cells can migrate during the repair process. (2) Inflammation—both neutrophils and monocytes are attracted to wound sites by a huge number of chemotaxis signals. The cells arrive at the wound site to clear the initial rush of contaminating bacteria and other pathogens. Meanwhile, neutrophils also secrete proinflammatory cytokines such as interleukin 1 alpha and beta (IL-1α and 1β) and tumor necrosis factor alpha (TNF-α). These then activate a cascade of downstream immune responses, and stimulate epidermal cells to proliferate. (3) Proliferation or reepithelialization—keratinocytes in the epidermal layer and fibroblasts in the dermal layer will start to proliferate and migrate toward the wound bed in response to the exposed extracellular matrix (ECM). Granulation tissue forms and the reepithelialization process is activated. (4) Tissue remodeling—after coverage of the wound bed, the remodeling process is activated. It consists of decreased ECM content and optimization of tissue structures. Specific to bone fractures, they usually need a longer time to heal (6 12 weeks). The healing can be divided into three major phases: reactive phase, reparative phase, and remodeling phase. (1) Reactive phase—This lasts 7 14 days and could be subdivided into fracture and inflammatory phase, and granulation tissue formation. (2) Reparative phase—This includes cartilage callus formation and mineral bone deposition. (3) Remodeling phase—This process substitutes the trabecular bone with compact bone. In adults, the strength of the healing bone is usually 80% of normal by 3 months after the injury.

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In contrast to normal wound healing, nonhealing chronic wounds, such as diabetic wounds, have special characteristics and challenges. (1) The structure of diabetic skin is in a pathological state. Under a microscope, the diabetic skin is seen to be thinner than that of normal skin. As well as the capillary density, collagen fibers and collagen content in the diabetic skin are significantly reduced. (2) Latent or superimposed microbial infections due to high blood glucose can cause the inflammation phase to be prolonged. Diabetic patients tolerate infection poorly and infection further affects blood glucose control. Hyperglycemia and inflammation affect each other and lead to a repetitive cycle. (3) Defective wound closure function is observed. The high level of glucose can affect keratinocytes in the epidermis and fibroblasts in the dermis directly. In addition, stem cells, such as epidermal stem cells, adipose-derived stem cells, and endothelial progenitor cells are also affected in both structure and biological functions. (4) Furthermore, hyperglycemia and the presence of an open wound create a negative nitrogen balance and lead to metabolic dysfunction. All these can impair the synthesis of proteins and collagen. Another wound-healing problem one often faces in clinical practice is the keloid. In contrast to the fetus where there is generally no sign of connective tissue scar formation after healing, all adults’ wounds heal by leaving a scar. Explanations could be due to the difference in transforming growth factor (TGF)-β1 expression, which is much higher in adults [12]. Thus, in a group of patients where there is a genetic predisposition to high TGF-β1 production, scar tissues can grow out of proportion in relation to the original wound—the formation of “keloid.” A thorough understanding of the mechanism of keloid formation is vital to designing nanomedicines to achieve better cosmesis.

9.4 The Mechanism and Advantages of Bionanomaterials in Wound Healing The mechanisms of bionanomaterials in promoting wound healing include antibacterial, antiinflammatory, regulating ECM production, promoting stem cell proliferation and differentiation, and enhancing growth factors (Fig. 9 3).

9.4.1 Antibacterial and Antiinflammatory Silver nitrate was used as ago as in the Second World War as an effective antibacterial drug. Currently, another silver compound, silver sulfadiazine is still used for burn wound treatment around the world. With advances in nanotechnology, the superior efficacy of AgNPs in bacterial killing has attracted the interest of researchers. AgNPs are much more efficient than other silver compounds in activity because of their larger surface area-to-volume ratio. The other reason for their significant antibacterial activity is the ability of AgNPs to damage the respiratory chain of mitochondria of bacteria and lead to bacteria cell death. In clinical practice, we have seen a rise in the number and percentage of infections caused by resistant bacteria. The fact that resistance to AgNPs is very low makes the use of this nanoparticle clinically an attractive

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FIGURE 9–3 The mechanism and application of bionanomaterials promoting wound healing.

option. Other envisaged potential applications for AgNPs would include in situ synthesis of AgNPs on polyester fabrics using NaOH/nano TiO2, the AgNPs carried by genipin crosslinked chitosan hydrogels, and textile materials based on TiO2 nanoparticles, which have selfcleaning and antibacterial properties [13 15]. Bionanomaterials can also have a strong antiinflammatory effect during the woundhealing process. Indeed, many of the published results have focused on the use of AgNPs. The advantage of AgNPs over silver salts is not only due to increased antimicrobial activity but also its antiinflammatory properties. The biological mechanisms of the antiinflammatory action of AgNPs may include: (1) effective bacterial killing by AgNPs will reduce the release of inflammatory agents such as lipopolysaccharides and free radicals and oxidative stress [16]; (2) modulation of cytokines at the wound site by AgNPs. Our group found IL-6 messenger RNA (mRNA) levels in wound areas treated with AgNPs were maintained at significantly lower levels throughout the healing process. In contrast, IL-10, and vascular endothelial growth factor (VEGF) mRNA in the AgNP group were higher than the control group at all time points [17]. Furthermore, we found that AgNPs might exert antiinflammatory activities by decreasing Interferon (IFN)-ɣ and TNF-α production [16,18]. When we conjugated AgNPs to dendrimer to form silver dendrimer nanocomposites, we demonstrated enhanced antiinflammatory activity compared with AgNPs or dendrimer alone [19].

9.4.2 Bionanomaterials Can Promote Wound Healing Due to Extracellular Matrix Regulation Directly As wound healing involves reepithelialization and wound contraction, our group observed the cellular response and events including epidermal reepithelialization and dermal contraction during wound healing. We found that AgNPs could drive fibroblast differentiation into

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myofibroblasts to promote wound contraction and thus increased the rate of wound closure [20]. It is well known that ECM is very important for cell proliferation both in vivo and in vitro. The main content of native ECM is collagen scaffold, and collagen is produced mainly by fibroblasts in the dermal layer. Types I and type III collagen are the most abundant. Collagen is essential in maintaining mechanical integrity, and it plays an important role in load bearing. Hence, it acts as protection against external stimuli and provides a platform for reepithelization. Our group explored the tensile properties of healed skin after treatment with AgNPs. Collagen fibrils can be seen clearly on scanning electronic microscopy in normal skin and in AgNP-treated healed skin. AgNPs improved tensile properties and led to better fibril alignments in repaired skin, with a close resemblance to normal skin [21]. More importantly, bionanomaterial-based tissue regeneration scaffolds provide new opportunities to mimic the natural intelligence and response of biological systems. For example, electrospinning nanofibrous structures were demonstrated to be morphologically close to natural ECM and could modulate cell behavior, and affect the efficiency of regeneration in biological tissues. It was found that electrospinning nanofibrous scaffolds could support attachment, spreading, and proliferation of mesenchymal stem cells (MSCs) [22]. Thus, nanofiber matrices can be used as scaffolds for soft tissue regeneration, such as in skin and skeletal muscle. Another study reported the use of synthetic, functional, and biodegradable peptide nanofiber gels for the improved healing of burn wounds. These bioactive nanofiber gels formed scaffolds that recapitulated the structure and function of the native ECM through signaling peptide epitopes, which could trigger angiogenesis through their affinity to basic growth factors [23]. These events have significant implications for wound treatment in clinical practice. Koutsopoulos et al. also reported the formation of multilayered three-dimensional (3D) tissues using peptide nanostructures. The cells proliferated continuously in these 3D constructs and deposited new ECM. This method has large potential to form functional skin tissues composed of multiple cell types and scaffold, as well as to understand cell biology in the 3D environment [24]. The assembled scaffolds closely mimic native ECM, with the scaffold’s fibers and pores being much smaller than cells. The scaffolds can be further modified and functionalized to enhance their interaction with other cells and tissues [25]. Romano et al. described how to synthesize protein and use multiple repeats of nanoscale peptide together and design full-length engineered ECM mimics. These were the ultimate goal for biological studies of cell matrix interactions, both in the physiological processes and in regenerative medicine. Another similar idea reported was multifunctionalized electrospinning nanofibrous scaffolds blended with mussel adhesive protein and polycaprolactone, which could serve as possible artificial skin [26].

9.4.3 Bionanomaterials Can Support Skin Regeneration by Promoting Stem Cell Growth Stem cells in skin play an important role in repairing the epidermis, regenerating hair, and maintaining tissue homeostasis. Hair follicle stem cells become active after wounding.

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In order to harness the potential of stem cells in tissue regeneration, the use of MSCs has been seen as a promising channel, due to easy isolation and culture as well as the expansion capacity. The availability of MSCs is relatively high since they can be found in bone marrow, adipose, and muscle tissue. In contrast to other stem cells, MSCs are also able to expand independent of the donor age [27]. We found AgNPs at a low concentration were able to promote MSC proliferation and increase their response to differentiation factors into an osteogenic lineage. Although the mechanism is still yet to be understood, it gives us an insight into using AgNP treatment for bone fractures [28]. It has been reported that AgNPs could enter human MSCs, combine with DNA, and activate the expression of genes such as hypoxia inducible factor-1ɑ (HIF-1ɑ) and IL-8, which in turn enhance cell proliferation [29,30]. In recent years, nanofibrous scaffolds coupled with stem cells are emerging as a key technique in the development of tissue engineering. Researchers have shown improved human cell growth on titanium dental implants through the formation of a nano-network surface oxide layer. They found the formation of a TiO2 nano-network on titanium surfaces could promote human MSC growth both in vitro and in vivo [31]. Grumezescu et al. evaluated the activity of wound dressing based on anionic polymers and magnetic nanoparticles loaded with usnic acid (Fe3O4@UA). They determined that the nano dressings could increase normal human fetal stem cell and antimicrobial properties [32].

9.4.4 Bionanomaterials Can Modulate Growth Factors in the Wound Site The three key components of tissue regeneration are scaffold, stem cells, and growth factors. Some growth factors play critical roles in the wound-healing process. Indeed, TGF-β1 has emerged as a major modulator of wound healing. One study showed that silver nanoparticle/ chitosan oligosaccharide/poly(vinyl alcohol) nanofibers markedly promoted fibroblast proliferation, collagen synthesis, and cell adherence. Importantly, the factors associated with the TGF-β1/Smad signal transduction pathway, such as TGF-β1, TGFβRI, TGFβRII, pSmad2, pSmad3, collagen I, collagen III, and fibronectin were also upregulated. Moreover, this enhancing effect was markedly inhibited by the TGFβRI receptor inhibitor, SB431542 [33,34]. Another problem of trying to use growth factors in healing is how to maintain their stability in wounds. Fibroblast growth factor (FGF) is a bioactive signaling molecule that stimulates cell proliferation and wound healing. A novel liposome with hydrogel core of liposomal silk fibroin (SF-LIP) has been successfully developed by the common liposomal template, followed by gelation of liquid SF inside a vesicle under sonication. SF-LIP is capable of encapsulating basic FGF (SF-bFGF-LIP) with high efficiency. SF-LIP effectively improves the stability of bFGF in wound fluids, and is very helpful in inducing regeneration of vascular vessels. Thus, SF-LIP may be a potential carrier for growth factors for wound healing [35]. Ogawa also presented nano-β-tricalcium phosphate scaffolds containing FGF-2 for use in periodontal tissue engineering [36]. In addition, many other reports have shown that human vascular endothelial growth factor [52,58], recombinant human epidermal growth factor [37 39], HIF-1α [40], and epidermal growth factor [41,42] could all be modulated by bionanomaterials during wound

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healing. Therefore, the nanofibrous system can be used not only as a skin cover, but also in broader applications in the field of regenerative medicine.

9.5 Potential Scope of Bionanomaterials in Wound Healing in Clinical Practice The scope of bionanomaterials in wound healing is wide and they have been used in skin wounds, bone fractures, vascular and neuron regeneration, and hemostasis.

9.5.1 The Application on Skin Wound Healing Skin is the largest organ of the human body. Normal skin consists of two layers: a keratinized stratified epidermis attached to a carpet of specialized matrix, and an underlying thicker layer of collagen-rich dermal connective tissue providing support and nourishment. The appendages, such as hair and glands, are derived from the epidermis but project deep into the dermal layer. Because the skin serves as a protective barrier against the outside world, any break in it must be rapidly and efficiently repaired. As discussed previously, inflammation plays a significant part in wound healing but this process must be controlled, with too little or too much inflammation both resulting in delayed healing. Indeed, in our ongoing study, we investigated how the duration of inflammation affected burn wound healing in the skin. It was found that although topical application of AgNPs immediately after burn injury significantly suppressed early inflammation, this resulted in delayed healing. In contrast, when AgNPs were given on post-burn day 3, which allowed the initiation of early inflammation in a controlled manner, faster burn wound healing was seen.

9.5.1.1 Application of Bionanomaterials in Dressings In general, an ideal wound dressing should be able to provide a moist wound environment, to protect the wound from secondary infections, to remove wound exudate, and to promote tissue regeneration. Specific additives with special functions can be introduced in advanced wound dressings with the aim of absorbing odor, providing strong antibacterial properties, smoothing pain, and relieving irritation. Because of the unique properties of a high ratio of surface area to volume, nanoscale size and porosity, nanofibers are used in wound dressings for wound care and management. To date, much progress has been made through the use of bionanomaterials in wound healing due to the ability of such materials to mimic the natural dimensions of tissue. Silver, mostly in the compound form of nitrate or sulfadiazine, is a well-studied antimicrobial agent and is commonly used in wound treatment. In comparison to antibiotics, there were early concerns of toxicity of silver. However, this is not specific and, as the doses used are very low, these concerns seem to be overstated. These types of dressings are currently applied in treatment of first- and second-degree burns. Furthermore, treatment of chronic wounds with dressings containing silver has also been shown to have significantly reduced bacterial load. Indeed, a research group has

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described a wound dressing composed of chitosan, hyaluronic acid, and AgNPs for diabetic foot ulcers. Their results suggested that the compound dressing could be used in diabetic foot ulcers even with the presence of antibiotic-resistant bacteria [43]. Another product was a nanofibrous membrane containing AgNPs and surface-grafted collagen. After modification, the nanofibrous membrane inhibited bacterial growth with a concomitant increase of membrane water absorption. This membrane was better than the commercial collagen sponge wound dressings [44]. Similarly, a dressing containing TiO2 nanoparticles was shown to accelerate healing of open excision type wounds in vivo and in vitro [45]. Kumar et al. developed chitin hydrogel nano ZnO composite bandages. The homogenized mixture of chitin hydrogel and nano ZnO was freeze-dried to obtain microporous composite bandages. The nanocomposite bandages showed enhanced swelling, blood clotting, and antibacterial activity. In addition, they reported the flexible and microporous chitosan hydrogel/nZnO bandages (CZBs) were also helpful in healing of nonhealing wounds. The manufactured CZBs with interconnected pores were shown to be helpful with regard to absorbing large volumes of wound exudates. CZBs were also demonstrated to have properties of controlled degradation, enhanced blood clotting, and excellent platelet activation [46,47]. Nitrocellulose liquid bandage is extensively used in hard-to-cover cut and wound management, owing to its flexibility, softness, transparency, and conformability [48]. The latest study was of new bioactive gelatin-oxidized starch nanofibers containing Lawsonia inermis (henna) for treating second-degree burn wounds. The dressing could enhance fibroblast attachment, proliferation, collagen secretion, and have antibacterial activity, with resultant accelerated wound closure [49]. Apart from providing the role of the shell, dressing fabrication using bionanomaterials can also be a carrier of growth factors or cytokines to promote wound healing. Chu et al. utilized a modified double-emulsion method with poly(lactic-co-glycolic acid) as the carrier to prepare recombinant human epidermal growth factor (rhEGF) nanoparticles. In fullthickness wound models in diabetic rats, rhEGF nanoparticles promoted the highest level of fibroblast proliferation, and this group of rats showed the fastest healing rate [37]. Similar results were seen in another experiment using chitosan-based hydrogel as a carrier for rhEGF nanoparticles [38,39]. Fabrication of bionanomaterials such as nanofibers can now be assisted by techniques like electrospinning. Nanofibrous membranes produced by electrospinning mimic the 3D structure of the ECM. Thus, nanofibrous dressings are a promising alternative for chronic wound healing, since they can act as a substitute for the natural ECM until it is repaired [50]. A further example is from a study describing the development of a poly(lactic-co-glycolic acid) (PLGA) nanofibrous membrane that contains rhEGF and Aloe vera (AV) extract. Both of these can promote wound healing: EGF is a wound-healing mediator and AV stimulates the proliferation and activity of fibroblast [51]. Wound dressing materials with ability to hold moisture, with excellent antimicrobial ability, and continuous efficacy are needed in daily clinical practice. With the early knowledge of AgNPs’ prohealing properties, development of a commercially available nanocrystalline silver-coated dressing, named Acticoat (Smith and Nephew plc, London, United Kingdom),

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came into fruition for burn wound treatment. The dressing was further evaluated in clinical patients with chronic venous leg ulcers. Healing was associated with a reduction in wound bacteria and neutrophils, and thus inflammation. It was concluded that an AgNP-coated dressing had beneficial effects on protecting the wound sites from bacterial contamination and promoting wound healing [52]. Another clinical study reported the use of nanocrystalline silver in an acute surgical wound to prevent localized skin necrosis due to infection and avoid skin grafting as a secondary procedure [53]. Because of the reduction in the number of dressing changes, AgNP dressings are less expensive. Another commercially available product is a hydroactive dressing containing a nanooligosaccharide factor. Indeed, a randomized clinical trial showed good cost-effectiveness in treating vascular leg ulcers with this hydroactive dressing when compared with a control [54]. A dressing based on lipidocolloid technology (TLC) impregnated with nanooligosaccharide factor (NOSF) has been developed. TLC NOSF was made of carboxymethylcellulose particles spread in a petroleum gel network. As for the randomized clinical trial, including 14 French hospital departments, the management of diabetic foot ulcer was studied. The results suggested that TLC NOSF matrices could be a beneficial therapeutic strategy for diabetic wounds. The results indicated that using TLC NOSF dressings in routine wound management can reduce the healing time. In addition, the data also suggested that the earlier this dressing is used, the shorter the time to closure, whatever the severity and nature of the chronic wounds [55].

9.5.1.2 Application of Bionanomaterials in Suture Fabrication Sutures have been used in surgery for hundreds of years. However, being a foreign material in the body, sutures might contribute toward the development of surgical site infections. For this reason, some commercially available antibiotic-coated sutures are on the market for the prevention of surgical site infection [56]. Recent innovations have involved advances in the material design. These improvements have led to more controlled absorption or decreased biodegradation, faster skin closure, and fewer potential infections. We fabricated the AgNP-coated Vicryl (Ethicon, United States) suture via layer-by-layer deposition method and tested it both in vitro and in an intestinal anastomosis model in mice. Our results showed that AgNps could be immobilized and distributed evenly on the surface of the suture. The AgNP-coated suture also demonstrated better antibacterial efficacy in vitro and more antiinflammatory activity in vivo [57,58]. Other groups reported fabrication of AgNP-coated sutures with different methods, including dipping technique, in situ photochemical deposition, and electrospinning [59 61]. The results were very much similar to ours. Despite these findings, clinical trials are still needed for evaluation.

9.5.1.3 Use of Bionanomaterials and Keloid Wound healing is an incredibly complex biological process that ends with the formation of a thickened collagen-enriched tissue called scar. Cutaneous scars lack many functional structures of the skin such as hair follicles, sweat glands, and sebaceous glands. Scar

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formation is a pervasive factor in our daily lives. In the case of serious traumatic injury, large scars can create long-lasting complications due to contraction and poor tissue remodeling. When the production of collagen becomes uncontrolled during healing, the resultant scar grows beyond the original size of the wound and becomes keloid. In terms of impaired wound healing, enhanced expression of TGF-β mRNA has been found in keloids scars. In contrast, a lack of TGF-β has been demonstrated to result in scarless healing in a fetal wound model. Indeed, inhibitors of TGF-β have been shown to reduce inflammation and scarring. Cumulatively, these results suggest that TGF-β plays an important role in tissue fibrosis and postinjury scarring [62]. Other cytokines such as IL-6 and IFN-Ɣ may also be involved in hypertrophic and keloid scar formation in adults [63]. As already mentioned, fetal tissue heals without scar. Artificial ECM acting as scaffolds has already been synthesized. These can self-assemble from simple precursors and yet display the complex properties of fully functional biological ECM. It may thus be possible to create an artificial fetal nano-environment in healing wounds and prevent scarring entirely. This environment could be made by small nanomachines that deploy upon contact with human tissue and begin the repair of damaged structures to result in perfectly healed wounds. The bionanomaterial compound hyaluronic acid nanoemulsions, as the carrier loading 10,11-methylenedioxycamptothecin, showed desirable skin-permeable capacity across human keloid skin and the drug was transferred directly into keloid lesion and demonstrated the potential inhibition of keloid fibroblast [64]. Another example was the use of cationic lipid nanoparticles. These could be loaded with mothers against decapentaplegic, drosophila, homolog of 3 (SMAD3)-antisense oligonucleotides and internalized onto keloid fibroblasts without toxicity. The complexes inhibited SMAD3 and collagen type I, also significantly suppressed in keloid fibroblasts [65]. The above examples of bionanomaterials may lead to new ways of treating keloids.

9.5.2 Bionanomaterials in Promoting Bone Fracture and Tendon Healing Nanotechnology has also opened the door to bone tissue engineering in that it can stimulate and contribute the reconstruction of complex tissue architectures. In particular, biopolymers are suitable materials as bionanoparticles for clinical application due to their versatile traits, including biocompatibility, biodegradability, and low immunogenicity. As implantable materials, nanoscale surface features can be introduced to implant materials that alter the immune response to the material or reduce the ability of bacteria to colonize it. For example, nanostructure polytetrafluoroethylene has been demonstrated to be nonimmunogenic in vivo due to low protein adsorption and low macrophage adhesion. The conjecture has been made that reducing immune response to foreign material may be less dependent on surface chemistry than on the surface features of nanomaterials. Therefore, the potential for developing implantable materials with immunologically inert surface nanoarchitecture can be greatly expanded.

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Various nano-based scaffolds have been shown to act on bone matrix to promote recruitment of circulating stem cells, and induce proliferation and eventual differentiation into mature osteoblasts [66]. Song aimed to fabricate 3D-printed ceramic scaffolds composed of nano-biphasic calcium phosphate (BCP), polyvinyl alcohol (PVA), and platelet-rich fibrin (PRF) at a low temperature without the addition of toxic chemicals. These 3D-printed BCP/PVA/PRF scaffolds with desired shapes and internal structures and incorporated bioactive factors could enhance the repair of segmental bone defects [67]. One of the strategies for improving bone tissue engineering is to stimulate the osteogenic differentiation and boneforming properties of bone progenitor cells. Synthetic polymer materials have been fabricated into nanoscale structures in order to prepare the matrix environment so that seeded cells could be induced to proliferate and differentiate toward desired lineages. Based on this, researchers investigated the cellular effects of gold nanoparticles on the differentiation of MSCs and found that osteogenic differentiation was regulated by the mitogen-activated protein kinase (MAPK) pathway [68]. Furthermore, others investigated the effects of vertically aligned silicon nanowire array on the differentiation of MSCs and found that this microenvironment could provide stimulation of osteogenesis and chondrogenesis via a number of mechanosensitive pathways [69]. At the same time, our group showed the ability of AgNPs to drive the proliferation of MSCs toward osteogenic differentiation under the influence of osteogenic growth factors, and the eventual promotion of fracture healing in a mouse femoral fracture model [28]. Another example of the efficacy of nanomaterials is the incorporation of boron nanoparticles with scaffold to improve bone tissue repair. In this regard, mesoporous bioactive glass (MBG) scaffolds were regarded as potential bone regeneration materials with better bioactivity and drug-delivery ability. Wu and his group explored boron-containing MBG scaffolds and demonstrated better mesopore structure, higher surface area, and nanopore volume when compared to non-MBG. Moreover, the scaffolds could effectively control the release of boron ions and significantly improved the proliferation and bone-related gene expression of osteoblasts [70]. Biomedical cements are considered promising injection materials for bone repair and regeneration. The technique of cement made from MBG nanoparticles has been presented already. This nanopowder-derived cement exhibited high surface area and in simulated body fluid, produced apatite nanocrystallites with an ultrafine size of 10 nm. The ultrafine nanocement adsorbed proteins, in particular positively charged proteins, at substantial levels. Furthermore, the early bone-forming response of the nanocement, based on implantation in a rat calvarial bone defect, demonstrated signs of osteoinductivity along with excellent osteocondution and bone matrix formation [71]. Bone cement based on polymethylmetacrylate (PMMA) is the standard for the anchoring of artificial joints. Like all biomaterials, PMMA has an elevated risk of infection when implanted into the human body, when compared to using autologous tissue. Therefore, the loading of PMMA with AgNPs to reduce the infection rate has been postulated [72]. Further exciting results were reported from using an injectable nano-calcium phosphate cement with appropriate mechanical, physical, and degradation rate which could potentially be utilized for filling bone defects [73].

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As for tendon healing, even after surgical repair, this remains a slow and tedious process. Healing time is typically several weeks. There is no currently utilized method that could promote tendon healing. The hurdle comes from the formation of scar tissue, which leads to weakening of the healed tendon, and consequently it being prone to rerupture. In addition, due to low metabolism in mature tendons, healing is usually a slow process in the adult population. Clinically, the healing of achilles tendon usually takes 4 8 weeks but a full return to sport activities is only recommended after a long time span of 4 12 months. We found that AgNPs could facilitate tendon repair in a rat model, through increased proteoglycan and collagen synthesis, paving the way to potential clinical applications in the future. In vitro results indicated a stimulatory effect on tenocyte proliferation and collagen synthesis by AgNPs. Histology also showed that AgNPs promoted cell alignment and proteoglycan synthesis, with improved collagen deposition [74]. Taken together, bionanomaterials can now be made through their nanoarchitecture to resist infection, decrease the body’s inflammatory response, and become more highly integrated. Thus, what have until now been purely “passive” implants could become “active” and functional. This will have a tremendous impact in clinical practice.

9.5.3 Bionanomaterials in Promoting Neuron Repair The field of regenerative medicine research often includes the manipulation of stem cells by nanoparticles and nano-structured surfaces, as well as tissue engineering to treat tissue loss as a result of disease and trauma. The exciting applications of nanotechnology have also been reported in neuron regenerative medicine. This includes spinal cord regrowth and retina regeneration, and the minimization of stroke dysfunction through neuron repair. The use of bionanomaterials can support the reconstitution of healthy tissues. Nerve regeneration after spinal cord injuries remains suboptimal despite recent advances in the field. One major hurdle is the rapid clearance of drugs from the injury site, which greatly limits therapeutic outcomes. Nanofiber scaffolds represent a potential class of materials for enhancing nerve regeneration because of their biomimicking architecture. Liu et al. investigated the feasibility of incorporating neurotrophin-3 and chondroitinase onto electrospun collagen nanofibers for the treatment of spinal cord injuries. Their results showed accelerated nerve regeneration through the provision of topographical signals and multiple biochemical cues arising from both nanofibrous scaffolds and cytokines [75]. Another approach was demonstrated by Ellis-Behnke et al. Self-assembling peptides which could spontaneously form nanofibers were used to create a scaffold-like tissue-bridging structure. This was tested in the regeneration of axons in acute brain injury of young and adult hamsters, with subsequent return of functional behavior [76].

9.5.4 The Bionanomaterials Promoting Healing in the Abdomen Abdominal trauma and surgical operations are common situations faced by surgeons. In this respect, our group explored the effect of AgNP-coated sutures on an intestinal anastomosis model and observed that this suture could improve anastomosis healing [57,58].

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Hemostasis can be a major problem during surgical procedures or after major trauma. Bleeding may result in a vicious, self-perpetuating cycle leading to severe physiologic derangements and multiorgan failure unless interrupted by effective treatment. Normal methods to stop bleeding include applying pressure, cauterization, and blood vessel ligation. Bionanomaterials could bring a new treatment modality. Ellis-Behnke used a self-assembling peptide that could form a nanofiber barrier to achieve complete hemostasis immediately when applied directly to cut liver wounds in animal study, without the use of pressure, cauterization, vasoconstriction, coagulation, or crosslinked adhesives. The self-assembling solution was nontoxic and nonimmunogenic, and the breakdown products are amino acids, which are tissue-building blocks that can be used to repair the site of injury. This first use of nanotechnology to achieve complete rapid hemostasis could fundamentally change how much blood is needed during surgery of the future [77]. On the other hand, Shakhssalim found that electrospinning poly(ε-caprolactone)/poly(L-lactic acid) scaffold could be a supportive substrate for bladder wall regeneration when seeded with bladder smooth muscle cells [78]. This bionanomaterial could be useful in patients who have had bladder excision for cancer. The bionanomaterials applied in abdomen injury need more attention and more experimental data to support their use.

9.6 Obstacles to Bionanomaterial Application Like any other drugs, the potential toxicity of bionanomaterials should be considered with great attention. Because of their small size, novel and unpredictable physicochemical properties are seen. Moreover, compared with conventional materials, nanoparticles can gain easy access to cells, tissues, even organs. It is well known that large nanoparticles (200 nm and above) are more efficient at activating the human complement system and are hence cleared faster from the blood by Kupffer cells than their smaller counterparts. Bionanomaterials may also have properties that could cause hazards to humans and the environment. Therefore, it is possible that the use of bionanomaterials could be a double-edged sword and they may affect the equilibrium of human health. Indeed, many researchers are already engaging in the evaluation of potential cytotoxicity of nanomaterials [79,80]. Most of these studies are in vitro toxicity studies, and the general consensus is that bionanomaterials at low dose will not cause significant cytotoxicity. However, just like most other agents or drugs, increasing concentration or exposure time of bionanomaterials will result in observable cytotoxicity. Furthermore, the toxicity thresholds for various cell types are also different. However, modifying the surface properties, as in the case of AgNPs, might prevent or limit their dispersion in solutions such as cell culture media, leading to reduced contact with the cell membrane and, therefore, preventing or limiting their uptake into cells [81]. For toxicology detection, the traditional experimental methods are not completely suitable to evaluate the risk of bionanomaterials, due to the small size effect and surface effect. Therefore, the future of nanomedicine will depend on the rational design of

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nanomaterials and tools based around a detailed and thorough understanding of biological processes rather than forcing applications for some materials currently in vogue. For example, through precise modulation of size and shape of bionanomaterials, the toxic effect will be modified. Furthermore, with the development of tissue engineering and scaffolds as well as a nanodelivery system, the sustained release of bionanomaterials in specifically targeted organs in the body can be achieved, thus reducing systemic toxicity. Nonetheless, it is imperative that for a new nanoproduct to be introduced, vigorous testing needs to be conducted to ensure the safety of patients. In the next decade, newer materials, technologies, and methods will be emerging to promote further development of nanomedicine. Meanwhile, more research work in this field will make this subject more mature and eventually serve as a more effective tool for our healthcare system.

9.7 Outlook of Bionanomaterials for Wound Healing in the Future The study of bionanomaterials is a relatively new field of research. It is obvious that further questions and studies are still needed in order to translate basic research into clinical applications. Meanwhile, detailed insights at the molecular level into the pathophysiology of various kinds of wound healing are very much needed. Other questions to be answered include the optimal therapeutic concentration and methods of encapsulation and delivery. The clinical application of nanotechnology also requires a number of regulatory guidelines to ensure the appropriate use of new medical devices and drugs originating from nanoscience and keeping potential hazards as low as possible. Thus, significant milestones in tissue engineering will require multiple disciplines working together in close collaboration. Material scientists, immunologists and biologists, surgeons, computer scientists, and regulatory agencies will need to collaborate. New bionanomaterials, new nanotechnologies, and related methods are emerging daily and will lead to the rapid development of nanomedicine to the benefit of patients.

References [1] J. Du, X. Liu, Nano biomaterials: a promising therapeutic strategy for skin wound healing in diabetic populations? Pyrex J. Med. Med. Sci. 4 (5) (2017) 44 49. [2] X. Liu, K.K. Wong, Application of nanomedicine in wound healing, in: A. Nasir, A. Friedman, S. Wang (Eds.), Nanotechnology in Dermatology, Springer Science 1 Business Media, New York, 2013, pp. 221 229. [3] P. Lee, R. Zhang, V. Li, X. Liu, R.W. Sun, C.M. Che, et al., Enhancement of anticancer efficacy using modified lipophilic nanoparticle drug encapsulation, Int. J. Nanomed. 7 (2012) 731 737. [4] P.Y. Lee, K.K. Wong, Nanomedicine: a new frontier in cancer therapeutics, Curr. Drug Deliv. 8 (3) (2011) 245 253. [5] P. Lee, K.K. Wong, Lipophilic-formulated gold porphyrin nanoparticles for chemotherapy, Methods Mol. Biol. 991 (2013) 93 98.

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[6] L.V. Zhukova, J. Kiwi, V.V. Nikandrov, TiO2 nanoparticles suppress Escherichia coli cell division in the absence of UV irradiation in acidic conditions, Colloids Surf. B Biointerfaces 97 (2012) 240 247. [7] M.S. Hassan, T. Amna, A. Mishra, et al., Fabrication, characterization and antibacterial effect of novel electrospinning TiO2 nanorods on a panel of pathogenic bacteria, J. Biomed. Nanotechnol. 8 (2012) 394 404. [8] F. Oyarzun-Ampuero, A. Vidal, M. Concha, J. Morales, S. Orellana, I. Moreno-Villoslada, Nanoparticles for the treatment of wounds, Curr. Pharm. Des. 21 (29) (2015) 4329 4341. [9] Y.I. Heit, P. Dastouri, D.L. Helm, G. Pietramaggiori, G. Younan, P. Erba, et al., Foam pore size is a critical interface parameter of suction-based wound healing devices, Plast. Reconstr. Surg. 129 (3) (2012) 589 597. [10] E.H. Zhou, C. Watson, R. Pizzo, J. Cohen, Q. Dang, P.M. Ferreira de Barros, et al., Assessing the impact of engineered nanoparticles on wound healing using a novel in vitro bioassay, Nanomedicine (Lond.) 9 (18) (2014) 2803 2815. [11] L.K. El-Gabry, O.G. Allam, O.A. Hakeim, Surface functionalization of viscose and polyester fabrics toward antibacterial and coloration properties, Carbohydr. Polym. 92 (1) (2013) 353 359. [12] P. Martin, Wound healing-aiming for perfect skin regeneration, Science 276 (4) (1997) 75 81. [13] P.J. Rivero, A. Urrutia, J. Goicoechea, F.J. Arregui, Bionanomaterials for functional textiles and fibers, Nanoscale Res. Lett. 10 (1) (2015) 501. [14] M. Montazer, S. Seifollahzadeh, Enhanced self-cleaning, antibacterial and UV protection properties of nano TiO2 treated textile through enzymatic pretreatment, Photochem. Photobiol. 87 (4) (2011) 877 883. [15] L. Gao, H. Gan, Z. Meng, R. Gu, Z. Wu, X. Zhu, et al., Evaluation of genipin-crosslinked chitosan hydrogels as a potential carrier for silver sulfadiazine nanocrystals, Colloids Surf. B Biointerfaces 148 (2016) 343 353. [16] K.K.Y. Wong, X.L. Liu, Silver nanoparticles—the real “silver bullet” in clinical medicine? Med. Chem. Commun. 1 (2010) 125 131. [17] J. Tian, K.K.Y. Wong, C.M. Ho, et al., Topical delivery of silver nanoparticles promotes wound healing, Chem. Med. Chem. 2 (2007) 129 136. [18] K.K.Y. Wong, S.O. Cheung, L. Huang, et al., Further evidence of the anti-inflammatory effects of silver nanoparticles, Chem. Med. Chem. 4 (2009) 1129 1135. [19] X. Liu, W. Hao, C.N. Lok, Y.C. Wang, R. Zhang, K.K. Wong, Dendrimer encapsulation enhances antiinflammatory efficacy of silver nanoparticles, J. Pediatr. Surg. 49 (12) (2014) 1846 1851. [20] X. Liu, P.Y. Lee, C.M. Ho, et al., Silver nanoparticles mediate differential responses in keratinocytes and fibroblasts during skin wound healing, Chem. Med. Chem. 5 (2010) 468 475. [21] K.H. Kwan, X. Liu, M.K. To, et al., Modulation of collagen alignment by silver nanoparticles results in better mechanical properties in wound healing, Nanomedicine 7 (2011) 497 504. [22] W. Ji, Y. Sun, F. Yang, J.J.J.P. van den Beucken, M. Fan, Z. Chen, et al., Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications, Pharm. Res. 28 (6) (2011) 1259 1272. [23] F. Yergoz, N. Hastar, C.E. Cimenci, A.D. Ozkan, T. Tekinay, M.O. Guler, et al., Heparin mimetic peptide nanofiber gel promotes regeneration of full thickness burn injury, Biomaterials 134 (2017) 117 127. [24] S. Koutsopoulos, L.D. Unsworth, Y. Nagai, S. Zhang, Controlled release of functional proteins through designer self-assembling peptide nanofiber hydrogel scaffold, Proc. Natl. Acad. Sci. U.S.A. 106 (12) (2009) 4623 4628. [25] F. Gelain, A. Lomander, A.L. Vescovi, S. Zhang, Systematic studies of a self-assembling peptidenanofiber scaffold with other scaffolds, J. Nanosci. Nanotechnol. 7 (2) (2007) 424 434.

Chapter 9 • Nanomaterials for Wound Healing: Scope and Advances

227

[26] B.J. Kim, H. Cheong, E.S. Choi, S.H. Yun, B.H. Choi, K.S. Park, et al., Accelerated skin wound healing using electrospinning nanofibrous mats blended with mussel adhesive protein and polycaprolactone, J. Biomed. Mater. Res. A 105 (1) (2017) 218 225. [27] P. Prager, M. Kunz, R. Ebert, L. Klein-Hitpass, J. Sieker, T. Barthel, et al., Mesenchymal stem cells isolated from the anterior cruciate ligament: characterization and comparison of cells from young and old donors, Knee Surg. Relat. Res. 30 (3) (2018) 193 205. [28] R. Zhang, P. Lee, V.C. Lui, Y. Chen, X. Liu, C.N. Lok, et al., Silver nanoparticles promote osteogenesis of mesenchymal stem cells and improve bone fracture healing in osteogenesis mechanism mouse model, Nanomedicine 11 (8) (2015) 1949 1959. [29] S.K. Jung, J.H. Kim, H.J. Kim, Y.H. Ji, J.H. Kim, S.W. Son, Silver nanoparticle-induced hMSC proliferation is associated with HIF-1ɑ-mediated upregulation of IL-8 expression, J. Invest. Dermatol. 134 (12) (2014) 3003 3007. [30] C. Greulich, S. Kittler, M. Epple, G. Muhr, M. Koller, Studies on the biocompatibility and the interaction of silver nanoparticles with human mesenchymal stem cells (hMSCs), Langenbecks Arch. Surg. 394 (3) (2009) 495 502. [31] C.Y. Chiang, S.H. Chiou, W.E. Yang, et al., Formation of TiO(2) nano-network on titanium surface increases the human cell growth, Dent. Mater. 25 (2009) 1022 1029. [32] A.M. Grumezescu, A.M. Holban, E. Andronescu, et al., Anionic polymers and 10 nm Fe3O4@UA wound dressings support human foetal stem cells normal development and exhibit great antimicrobial properties, Int. J. Pharm. 463 (2014) 146 154. [33] L. Zi-Wei, C.W. Li, Q. Wang, S.J. Shi, M. Hu, Q. Zhang, et al., The cellular and molecular mechanisms underlying silver nanoparticle/chitosan oligosaccharide/poly(vinyl alcohol) nanofiber-mediated wound healing, J. Biomed. Nanotechnol. 13 (1) (2017) 17 34. [34] C.W. Li, Q. Wang, J. Li, M. Hu, S.J. Shi, Z.W. Li, et al., Silver nanoparticles/chitosan oligosaccharide/poly (vinyl alcohol) nanofiber promotes wound healing by activating TGFβ1/Smad signaling pathway, Int. J. Nanomed. 11 (2016) 373 386. [35] H.L. Xu, P.P. Chen, D.L. ZhuGe, Q.Y. Zhu, B.H. Jin, B.X. Shen, et al., Liposomes with silk fibroin hydrogel core to stabilize bFGF and promote the wound healing of mice with deep second-degree scald, Adv. Healthc. Mater. 6 (19) (2017). Epub 2017 June 29. [36] K. Ogawa, H. Miyaji, A. Kato, Y. Kosen, T. Momose, T. Yoshida, et al., Periodontal tissue engineering by nano beta-tricalcium phosphate scaffold and fibroblast growth factor-2 in one-wall infrabony defects of dogs, J. Periodontal. Res. 51 (6) (2016) 758 767. [37] Y. Chu, D. Yu, P. Wang, J. Xu, D. Li, M. Ding, Nanotechnology promotes the full-thickness diabetic wound healing effect of recombinant human epidermal growth factor in diabetic rats, Wound Repair Regen. 18 (5) (2010) 499 505. [38] M. Hajimiri, S. Shahverdi, M.A. Esfandiari, B. Larijani, F. Atyabi, A. Rajabiani, et al., Preparation of hydrogel embedded polymer-growth factor conjugated nanoparticles as a diabetic wound dressing, Drug. Dev. Ind. Pharm. 42 (5) (2016) 707 719. [39] K. Vocetkova, M. Buzgo, V. Sovkova, D. Bezdekova, P. Kneppo, E. Amler, Nanofibrous polycaprolactone scaffolds with adhered platelets stimulate proliferation of skin cells, Cell Prolif. 49 (5) (2016) 568 578. [40] Y. Heun, K. Pogoda, M. Anton, J. Pircher, A. Pfeifer, M. Woernle, et al., HIF-1ɑ dependent wound healing angiogenesis in vivo can be controlled by site-specific lentiviral magnetic targeting of SHP-2, Mol. Ther. 25 (7) (2017) 1616 1627. [41] X. Li, X. Ye, J. Qi, R. Fan, X. Gao, Y. Wu, et al., EGF and curcumin co-encapsulated nanoparticle/hydrogel system as potent skin regeneration agent, Int. J. Nanomed. 11 (2016) 3993 4009. [42] M. Rizzi, M. Migliario, V. Rocchetti, S. Tonello, Epiregulin-loaded PLGA nanoparticles increase human keratinocytes proliferation: preliminary data, Eur. Rev. Med. Pharmacol. Sci. 20 (12) (2016) 2484 2490.

228

THERANOSTIC BIONANOMATERIALS

[43] B.S. Anisha, R. Biswas, K.P. Chennazhi, R. Jayakumar, Chitosan-hyaluronic acid/nano silver composite sponges for drug resistant bacteria infected diabetic wounds, Int. J. Biol. Macromol. 62 (2013) 310 320. [44] J. Bao, B. Yang, Y. Sun, Y. Zu, Y. Deng, A berberine-loaded electrospinning poly-(epsilon-caprolactone) nanofibrous membrane with hemostatic potential and antimicrobial property for wound dressing, J. Biomed. Nanotechnol. 9 (7) (2013) 1173 1180. [45] D. Archana, B.K. Singh, J. Dutta, P.K. Dutta, In vivo evaluation of chitosan-PVP-titanium dioxide nanocomposite as wound dressing material, Carbohydr. Polym. 95 (1) (2013) 530 539. [46] P.T. Kumar, V.K. Lakshmanan, T.V. Anilkumar, C. Ramya, P. Reshmi, A.G. Unnikrishnan, et al., Flexible and microporous chitosan hydrogel/nano ZnO composite bandages for wound dressing: in vitro and in vivo evaluation, ACS Appl. Mater. Interfaces 4 (5) (2012) 2618 2629. [47] P.T. Kumar, V.K. Lakshmanan, R. Biswas, S.V. Nair, R. Jayakumar, Synthesis and biological evaluation of chitin hydrogel/nano ZnO composite bandage as antibacterial wound dressing, J. Biomed. Nanotechnol. 8 (6) (2012) 891 900. [48] X. Mu, H. Yu, C. Zhang, X. Chen, Z. Cheng, R. Bai, et al., Nano-porous nitrocellulose liquid bandage modulates cell and cytokine response and accelerates cutaneous wound healing in a mouse model, Carbohydr. Polym. 136 (2016) 618 629. [49] Z. Hadisi, J. Nourmohammadi, S.M. Nassiri, The antibacterial and anti-inflammatory investigation of Lawsonia inermis-gelatin-starch nano-fibrous dressing in burn wound, Int. J. Biol. Macromol. 107 (Pt B) (2018) 2008 2019. Å ˇ [50] E. Adomaviˇciut˙e, S. Stanys, M. Zilius, V. Juˇskait˙e, A. Pavilonis, V. Briedis, Formation and biopharmaceutical characterization of electrospinning PVP mats with propolis and silver nanoparticles for fast releasing wound dressing, Biomed. Res. Int. 2016 (2016) 4648287. [51] I. Garcia-Orue, G. Gainza, F.B. Gutierrez, J.J. Aguirre, C. Evora, J.L. Pedraz, et al., Novel nanofibrous dressings containing rhEGF and aloe vera for wound healing applications, Int. J. Pharm. 523 (2) (2017) 556 566. [52] R.G. Sibbald, J. Contreras-Ruiz, P. Coutts, M. Fierheller, A. Rothman, K. Woo, Bacteriology, inflammation, and healing: a study of nanocrystalline silver dressings in chronic venous leg ulcers, Adv. Skin Wound Care 20 (10) (2007) 549 558. [53] M. Bhattacharyya, H. Bradley, A case report of the use of nanocrystalline silver dressing in the management of acute surgical site wound infected with MRSA to prevent cutaneous necrosis following revision surgery, Int. J. Low Extrem. Wounds 7 (1) (2008) 45 48. [54] M. Augustin, K. Herberger, K. Kroeger, K.C. Muenter, L. Goepel, R. Rychlik, Cost-effectiveness of treating vascular leg ulcers with UrgoStart(s) and UrgoCell(s) contact, Int. Wound J. 13 (1) (2016) 82 87. [55] D.R. Shanahan, The explorer study: the first double-blind RCT to assess the efficacy of TLC NOSF on DFUs, J. Wound Care 22 (2) (2013) 78 82. [56] S. Takeno, K. Yamashita, Y. Yamashita, K. Yamada, S. Hoshino, Y. Yamauchi, et al., The results of a propensity score matching analysis of the efficacy of abdominal fascia and skin closure using PDS@ plus antibacterial (polydioxanone) sutures on the incidence of superficial incisional surgical site infections after gastroenterologic surgery, Surg. Infect. (Larchmt) 17 (1) (2016) 94 99. [57] S. Zhang, X. Liu, H. Wang, J. Peng, K.K. Wong, Silver nanoparticle-coated suture effectively reduces inflammation and improves mechanical strength at intestinal anastomosis in mice, J. Pediatr. Surg. 49 (4) (2014) 606 613. [58] X. Liu, P. Gao, J. Du, X. Zhao, K.K. Wong, Long-term anti-inflammatory efficacy in intestinal anastomosis in mice using silver nanoparticle-coated suture, J. Pediatr. Surg. 52 (12) (2017) 2083 2087. [59] S. Chen, L. Ge, A. Mueller, M.A. Carlson, M.J. Teusink, F.D. Shuler, et al., Twisting electrospinning nanofiber fine strips into functional sutures for sustained co-delivery of gentamicin and silver, Nanomedicine 13 (4) (2017) 1435 1445.

Chapter 9 • Nanomaterials for Wound Healing: Scope and Advances

229

[60] A.L. Gallo, F. Paladini, A. Romano, T. Verri, A. Quattrini, A. Sannino, et al., Efficacy of silver coated surgical sutures on bacterial contamination, cellular response and wound healing, Mater. Sci. Eng. C Mater. Biol. Appl. 69 (2016) 884 893. [61] S. De Simone, A.L. Gallo, F. Paladini, A. Sannino, M. Pollini, Development of silver nano-coatings on silk sutures as a novel approach against surgical infections, J. Mater. Sci. Mater. Med. 25 (9) (2014) 2205 2214. [62] P. Rorison, A. Thomlinson, Z. Hassan, S.A. Roberts, M.W. Ferguson, M. Shah, Longitudinal changes in plasma transforming growth factor beta-1 and post-burn scarring in children, Burns 36 (1) (2010) 89 96. [63] H. Xue, R.L. McCauley, W. Zhang, Elevated interleukin-6 expression in keloid fibroblasts, J. Surg. Res. 89 (1) (2000) 74 77. [64] Y. Gao, X. Cheng, Z. Wang, J. Wang, T. Gao, P. Li, et al., Transdermal delivery of 10,11-methylenedioxycamptothecin by hyaluronic acid based nanoemulsion for inhibition of keloid fibroblast, Carbohydr. Polym. 112 (2014) 376 386. [65] S.E. Jin, C.K. Kim, Y.B. Kim, Cellular delivery of cationic lipid nanoparticle-based SMAD3 antisense oligonucleotides for the inhibition of collagen production in keloid fibroblasts, Eur. J. Pharm. Biopharm. 82 (1) (2012) 19 26. [66] K.K.Y. Wong, X.L. Liu, Nanomedicine: a primer for surgeons, Pediatr. Surg. Int. 28 (2012) 943 951. [67] Y. Song, K. Lin, S. He, C. Wang, S. Zhang, D. Li, et al., Nano-biphasic calcium phosphate/polyvinyl alcohol composites with enhanced bioactivity for bone repair via low-temperature three-dimensional printing and loading with platelet-rich fibrin, Int. J. Nanomed. 13 (2018) 505 523. [68] C. Yi, D. Liu, C.C. Fong, et al., Gold nanoparticles promote osteogenic differentiation of mesenchymal stem cell through p38 MAPK pathway, ACS Nano 4 (11) (2010) 6439 6448. [69] D. Liu, C. Yi, C.C. Fong, et al., Activation of multiple signaling pathways during the differentiation of mesenchymal stem cells cultured in a silicon nanowire microenvironment, Nanomedicine: NBM 10 (2) (2014) 1153 1163. [70] C. Wu, R. Miron, A. Sculean, S. Kaskel, T. Doert, R. Schulze, et al., Proliferation, differentiation and gene expression of osteoblasts in boron-containing associated with dexamethasone deliver from mesoporous bioactive glass scaffolds, Biomaterials 32 (29) (2011) 7068 7078. [71] M.S. Kang, N.H. Lee, R.K. Singh, N. Mandakhbayar, R.A. Perez, J.H. Lee, et al., Nanocements produced from mesoporous bioactive glass nanoparticles, Biomaterials 162 (2018) 183 199. [72] V. Alt, T. Bechert, P. Steinrucke, et al., An in vitro assessment of the antibacterial properties and cytotoxicity of nanoparticulate silver bone cement, Biomaterials 25 (18) (2004) 4383 4391. [73] M. Roozbahani, M. Alehosseini, M. Kharaziha, R. Emadi, Nan-calcium phosphate bone cement based on Si-stabilized ɑan-calcium phosphate with improved mechanical properties, Mater. Sci. Eng. C Mater. Biol. Appl. 81 (2017) 532 541. [74] K.H. Kwan, K.W. Yeung, X. Liu, K.K. Wong, H.C. Shum, Y.W. Lam, et al., Silver nanoparticles alter proteoglycan expression in the promotion of tendon repair, Nanomedicine 10 (7) (2014) 1375 1383. [75] T. Liu, J. Xu, B.P. Chan, S.Y. Chew, Sustained release of neurotrophin-3 and chondroitinase ABC from electrospinning collagen nanofiber scaffold for spinal cord injury repair, J. Biomed. Mater. Res. A 100 (1) (2012) 236 242. [76] R.G. Ellis-Behnke, G.E. Schneider, Peptide amphiphiles and porous biodegradable scaffolds for tissue regeneration in the brain and spinal cord, Methods Mol. Biol. 726 (2011) 259 281. [77] R.G. Ellis-Behnke, Y.X. Liang, D.K. Tay, P.W. Kau, G.E. Schneider, S. Zhang, et al., Nano hemostat solution: immediate hemostasis at the nanoscale, Nanomedicine 2 (4) (2006) 207 215. [78] N. Shakhssalim, M. Soleimani, M.M. Dehghan, J. Rasouli, M. Taghizadeh-Jahed, P.M. Torbati, et al., Bladder smooth muscle cells on electrospinning poly(Ɛ-caprolactone)/poly(l-lactic acid) scaffold promote bladder regeneration in a canine model, Mater. Sci. Eng. C Mater. Biol. Appl. 75 (2017) 877 884.

230

THERANOSTIC BIONANOMATERIALS

[79] X. Chen, H.J. Schluesener, Nanosilver: a nanoproduct in medical application, Toxicol. Lett. 176 (1) (2008) 1 12. [80] P.K. Lam, E.S.Y. Chan, W.S. Ho, et al., In vitro cytotoxicity testing of a nanocrystalline silver dressing (Acticoat) on cultured keratinocytes, Br. J. Biomed. Sci. 61 (3) (2004) 125 127. [81] C. Carlson, S.M. Hussain, A.M. Schrand, et al., Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species, J. Phys. Chem. B 112 (43) (2008) 13608 13619.

10 Advanced Nanovaccines for Immunotherapy Applications: From Concept to Animal Tests Flavia Fontana, Patrícia Figueiredo, Hélder A. Santos DR UG RESEARCH PROGRAM, DIVI SION OF PHAR MACE UTICAL CHEMISTRY AND TECHNOLOGY, FACULTY OF PHARMACY, UNIVERSITY OF HELSINKI, HELSINKI, FINLAND

10.1 Introduction 10.1.1 Immunotherapy Our health status is the result of a delicate balance of forces within the immune system, that is, the interactions with the external world, the continuous surveillance to identify mutated cells, and the reaction to viral or bacterial infections are all controlled by a mighty army of immune cells [1]. Immunotherapy, thereby, defines any treatment targeted to the immune system with the aim of initiating, rechallenging, suppressing, or modulating its action [2]. The history of immunotherapy is quite long, dating back from the first attempts at vaccination to the recent popularity of monoclonal antibodies in cancer immunotherapy [3]. The main actors playing a role in the control center of the immune system are antigenpresenting cells (APCs), which bridge innate immunity to the adaptive one by capturing foreign and self-proteins and processing them into antigens, short (8 15 amino acids) peptides [4]. These antigens are then loaded onto proteins, major histocompatibility complexes (MHCs), to present them to naïve lymphocytes [5]. Endogenous antigens are presented on MHC class I, which activate CD81 T cells, while antigens derived from exogenous proteins are loaded onto MCH II to prime CD41 T cells [6]. In physiological conditions, APCs need additional stimuli to mature and initiate the immune response, in case of external dangers (e.g., bacteria and viruses), peptidic antigens are often mixed with other danger signal [e.g., lipopolysaccharide (LPS), bacterial and viral nucleic acids, like CpG sequences or single- and double-stranded RNA, and bacterial antigens like flagellin] that promote the activation of, among others, Toll-like receptor (TLR) [7]. However, sometimes, an infection is not detected directly through the pathogen, but by the release of intracellular self-molecules in the

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extracellular space; these molecules are identified as danger-associated molecular patterns (DAMPs) [8]. Among the DAMPs recognized by APCs are, for example, crystals of uric acid, presence of extracellular ATP, presence of oxidized phospholipids, and calreticulin [9]. These signals are also present in the case of cellular apoptosis, the immune reaction they initiate is correlated to the adjuvancy of the DAMPs released and the antigenicity of the peptides presented, because such a reaction has to overcome both the central and peripheral tolerance mechanisms [9]. These switches prevent the development of autoimmunity, by selecting clones of T cells not reacting with self-peptides [10,11]. However, in the case of autoimmune diseases, the presence of DAMPs (e.g., self-nucleic acids) baffles the peripheral tolerance, setting in motion a reaction against healthy cells presenting a self-peptide [12].The actual role in killing infected cells or producing antibodies against a pathogen is then left to lymphocytes, either cytotoxic T cells or B cells, with the help and support of CD41 helper T cells [2]. Immunotherapy employs small drug molecules, monoclonal antibodies, vaccines to interfere with the immunological processes happening within the patient to prevent, treat (controlling the symptoms), or cure the disease, as presented in Table 10 1. Vaccines against different pathogens have long been established, leading to the eradication of some viral diseases [13]. In spite of this, we still lack effective vaccines against other pathogens (e.g., HIV and Ebola) [14,15] or, to avoid multiple rechallenges, the potency of the vaccine could be improved [16]. Another challenging field for vaccine development concerns the therapeutic “vaccines” for cancer immunotherapy. Preventive cancer vaccines have been established for clinical use [e.g., vaccines for human papilloma virus (HPV)] [17], but clinical trials into therapeutic cancer vaccines have only produced a dendritic cell-based vaccine (Sipulcel-T) for the treatment of prostate cancer [18]. Monoclonal antibodies are employed clinically both in the treatment of autoimmune diseases and cancer by selectively targeting receptors, growth factors, immune modulators, as reported in Table 10 1. Finally, small drug molecules are either currently in use (e.g., steroids and methotrexate in the treatment of autoimmune diseases) or are in the discovery and development process (e.g., small-molecule inhibitors of immune checkpoint and modulators of the amino acid catabolism) [19,20]. The current therapies available for immunotherapy present, however, some disadvantages, including: (1) the immunogenicity of vaccines decreased in the years, due to progress in the production, with the preparation of more and more purified antigenic sources, requiring thereby multiple rechallenges to instaurate immune memory; and (2) more potent vaccines are needed also for the development of “therapeutic” cancer vaccines [34]. The use of monoclonal antibodies has greatly improved the prognosis of patients suffering from both autoimmune diseases or cancer, but, unfortunately, not all patients respond to the therapy, needing thereby the design of combinatorial schemes of treatments. Small drug molecules have shown efficacy in the treatment of autoimmunity, however, their action is often systemic, leading to off-target action and side effects. Finally, as for the small drug molecules currently developed for cancer immunotherapy, often they display unfavorable properties for their formulation and some degree of systemic toxicity.

Table 10–1 Current Immunotherapies Available as Vaccines, for Cancer Immunotherapy and for the Treatment of Autoimmune Diseases Treatment

Disease

Type of Drug

Mode of Action

Clinical Use

References

Influenza vaccine

Influenza

[21]

HPV-caused cervix cancer

• • • •

Yes

HPV vaccines

• Live inactivated • Trivalent inactivated Recombinant

Sipulcel-T

Castration-resistant prostate cancer Various cancer types

Therapeutic cancer vaccines

DC pulsed with tumor antigen and GM-CSF • DC-based vaccines • Peptide-based vaccines • Whole tumor cell lysate Monoclonal antibody

Alemtuzumab

Multiple sclerosis

Anti TNF-α

ICIs Monoclonal antibodies

Immune-mediated inflammatory diseases (e.g., rheumatoid arthritis) Different cancers Different cancers

Monoclonal antibodies Monoclonal antibodies

Glucocorticoids

Rheumatoid arthritis

Small drug molecules

Methotrexate

Rheumatoid arthritis

Small drug molecule

COX2 Inhibitors

Rheumatoid arthritis

Small drug molecule

Rapamycine

Immunosuppression in transplanted patients Different cancers

Small drug molecule Small drug molecules

Adenosine signaling receptor antagonists

Different cancers

Small drug molecules

CD39/CD73 Inhibitors TLR-agonists

Different cancers Different cancers

Small drug molecules Small drug molecules

“IDO” family inhibitors

Monoclonal antibody

Activation of APCs Production of antibodies (humoral) Activation of APCs Production of antibodies (humoral) Priming of T cells

Yes

[17,22,23]

Yes

[24]

Priming of T cells

No

[18,25]

• Blocking the CD52 receptor on lymphocytes • Pan-lympho depletion Binding to TNF-α

Yes

[26]

Yes Biosimilar developed

[27,28]

Yes Yes

[29] [30]

Yes

[31]

Yes

[32]

Yes

[20]

Yes

[33]

No, in clinical trials as first-line and as combo therapy No, research stage

[20]

No, research stage Yes, clinical trials

[20] [20]

Binding to CTLA-4, PD1 or PD-L1 Binding to different targets Antibody-dependent cell-mediated cytotoxicity Genomic and nongenomic interference on the transcription factors Not completely elucidated Potentiation of the adenosine signal PGE2 generation Induction of immunosuppression Binding to transcriptional factors with inhibition of cytokine production Inhibition of amino acid catabolism Decrease in cAMP Avoiding the blunting of the immune response Decreasing the production of adenosine Adjuvants

[20]

APC, Antigen-presenting cell; DC, dendritic cell; GM-CSF, granulocyte macrophage colony stimulating factor; HPV, human papilloma virus; ICIs, immune checkpoint inhibitors; TLR, Toll-like receptor.

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10.1.2 Nanotechnology for Immunotherapy Nanoparticles have retained a central place in the research stage in drug-delivery systems over the last few decades, due to the promising advantages over conventional formulations [35]. In particular, the delivery of drugs with nanoparticles enhances the efficacy of the drug, while lowering the side effects derived from a systemic administration, which allows the reevaluation of compounds discarded in the industrial pipeline due to their unfavorable properties for formulation [36]. Moreover, combinations of different drugs or of drugs and diagnostic (imaging) moieties (theranostics) can be achieved within one particle [37,38]. Further applications focus on nanomaterials for tissue engineering with the incorporation of nanoparticles in scaffolds for hard tissues, or the production of tissues featuring nanosized moieties [36]. Nanosystems have been widely investigated as immunotherapy and they have been proposed as innovative adjuvants for vaccines, as well as delivery systems for immunosuppressive drugs [39]. The main advantages derived from the use of nanotechnology in immunotherapy are the possibility to obtain size-dependent delivery to the lymphoid organs, the creation of antigenic depots that increase the immunogenicity of antigens, but may result in long-term exhaustion of the immune response, the possibility to modify the surface of the particles with antigenic or adjuvant moieties to mimic pathogens, and finally the uptake of nanoparticles by APCs increases the cross-presentation of antigens on MHC I, considered a useful feature for cancer immunotherapy [40,41]. Nanovaccines generally carry both antigens and adjuvants and are work themselves as adjuvants or induce immunogenic cell death with the release of tumor antigens [39]. These particles are internalized by APCs either at the site of injection or after trafficking to the lymph nodes, and they then activate the APC [34]. Activated APCs prime immunostimulatory T cells by the combination of three different signals, as presented in Fig. 10 1.

FIGURE 10–1 Immunostimulation process. Nanoparticles activate resting dendritic cells, leading to the priming of T cells through a combination of three signals: presentation of the antigen on MHC to the T-cell receptor, expression of costimulatory signals (e.g., CD80), and, finally, with the secretion of proinflammatory cytokines. (1) Presentation of the antigen on MHC for binding to the T-cell receptor; (2) presentation of costimulatory signals (e.g., CD80 and CD86); and (3) secretion of proinflammatory cytokines to create an inflammatory environment surrounding the T cell. MHC, Major histocompatibility complex. Part of the artworks in this figure is adapted from Servier SmartArt.

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FIGURE 10–2 Nanoparticle-mediated immunosuppression process. Immunoneutral nanoparticles interact with APCs, delivering immunosuppressive drugs, disease-related antigens, or a combination of both. APCs assume a tolerogenic phenotype: they present the antigen on MHC, but do not present costimulatory signals nor do they secrete proinflammatory cytokines. The interaction of such APCs with naïve T cells leads to the priming of antigenspecific regulatory T cells, while the interaction with autoreactive T cells leads to their anergy. APC, Antigenpresenting cell; MHC, major histocompatibility complex. Part of the artworks in this figure is adapted from Servier SmartArt.

As for the use of nanotechnology in autoimmune diseases, nanoparticles allow the targeted delivery of immunosuppressant with the administration of a higher dose, while reducing the systemic side effects. Moreover, the particles can be selectively targeted directly to the effector cells (T cells) responsible for the disease, neutralizing them. Finally, immunoneutral particles can deliver antigens to APCs in a “tolerogenic” way, leading to the priming of antigen-specific regulatory T cells (Fig. 10 2) [42]. In the next section, the parameters useful for a rational design of the nanosystem for either immunostimulation or immunosuppression are presented, followed by examples of in vitro assays for the preliminary screening of the formulations, and by the main in vivo models employed in cancer immunotherapy or as models of autoimmune diseases.

10.2 Design of the Systems Understanding the interactions between nanomaterials and immune cells is crucial when considering the use of nanoparticles (NPs) for immunotherapy applications. The physicochemical properties of NPs (e.g., size, shape, surface charge and chemistry, and ligand density) can be tuned and engineered to ease the biodistribution and therapeutic loading, site-specific targeting, and immunogenicity [39,43]. The parameters that should be taken into consideration when designing nanocarriers for immunotherapy are summarized in Fig. 10 3, and are be discussed in detail in the following subsections.

10.2.1 Size Among other physicochemical properties of NPs, size is one of the main factors influencing the cellular interaction and uptake of NPs, their blood circulation time, and biodistribution

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FIGURE 10–3 Different parameters for a rational design of nanosystems to enhance the delivery of immunotherapies, increase tissue permeation, and avoid nonspecific uptake.

in vivo [44,45]. Together with the size of NPs, the route of administration also affects the biodistribution of NPs. After intravenous administration, NPs larger than 200 nm tend to accumulate in the liver and spleen, while smaller NPs (,5 nm) rapidly undergo renal clearance [46,47]. Upon subcutaneous or intradermal administration, the design of the vaccine needs to take into consideration the transport of antigens from peripheral tissues to the secondary lymphoid organs via the lymphatic system. NPs smaller than 200 nm are able to directly reach the lymphatic organs through the lymphatic system, while NPs larger than 200 nm need to be endocytosed by dendritic cell (DCs) for transport to the lymph nodes, a process requiring c.24 h that might affect the amount of antigen reaching the lymphatic organs [48,49]. Moreover, ultrasmall NPs with a hydrodynamic diameter of 25 nm were taken up by the lymphatic capillary network more efficiently than NPs with 100 nm of hydrodynamic diameter, where c.50% compared to 6% of the DCs isolated from the lymph node contained NPs 24 h after injection [50].

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In addition to the cellular uptake of the antigen-containing NPs by APCs, a robust antigen presentation is crucial to induce potent cytotoxic T lymphocyte (CTL) responses. For that, exogenously delivered antigens need to be presented by DCs to the CD81 T cells via “crosspresentation” [51]. During the initial degradation in phagosomes, this process needs to be tightly controlled to avoid the degradation of immunogenic peptides, and thus, maximizing the recognition of the antigenic peptides by CD81 T cells [52]. The composition and activities of the lysosomal proteases and protein degradation can be modulated by the phagosomal pH [53]. Consequently, the size of the antigen-containing NPs will affect the intracellular trafficking of the antigen: the antigen prepared in particles larger than 200 nm will be transported to early endosomal compartments with a pH of 6.0, while particles smaller than 200 nm will be shuttled to late endosome/lysosome with a pH of 4.5 5.0. Antigen degradation is limited at more basic pH, resulting in higher efficiency in the cross-presentation process for larger particles than for smaller ones, and therefore enhanced CD81 T-cell activation [54,55].

10.2.2 Shape As well as size, the NPs’ shape also plays a role in the circulation time in the bloodstream, biodistribution, targeting delivery, and cellular uptake efficiency, as well as the immune response [47,56]. Recent developments in NP engineering have originated different shapes from sphere-shaped, including rods, prisms, branched structures, cubes, worms, and disks [57]. Here, the ratio between the height and width of the particle, that is, the aspect ratio (AR), is representative of the particle shape. Huang et al. prepared mesoporous silica NPs with similar particle diameter, chemical composition, and surface charge, but with different AR and lengths: 100 nm spherical (AR 5 1), 240 nm short rod (AR 5 2), and 450 nm long rod (AR 5 4) [58]. The internalization of the different mesoporous silica NPs by A375 cells was found to be dependent on the AR of the prepared particles, suggesting that larger AR particles were more easily taken up and presented a faster internalization rate, mostly due to the larger contact area with the cell membrane than the spherical NPs. In addition, the shape of the NPs also affects the margination dynamics or the lateral drift of NPs toward the endothelial walls (Fig. 10 4). Here, variable forces and torques applied on rod-shaped particles under flow allow them to marginate and drift toward the vessel walls in which their association with the endothelial cells favors particle 2 cell interactions and allows extravasation through the fenestrated vasculature of tumors [47]. However, the spherical particles travel in the bloodstream through the cell-free layer region of the vessel, exhibiting a slight lateral drift and less ability to establish contact/binding points with endothelial cells [59]. Apart from the influence on cellular uptake, the shape of NPs can also affect the modulation of the immune response. In a recent study, Kumar et al. analyzed the influence of diverse particular systems with different sizes and shapes on the antigen (chicken ovalbumin OVA) presentation and consequent processing by the immune cells [60]. The results showed a size- and shape-dependent modulation of immune responses, in which the small spherical NPs (193 nm in diameter) generated a Th1-biased response, while the rod-shaped particles (1530 nm in length) stimulated a Th2-biased response against OVA.

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FIGURE 10–4 (A) Influence of the particle shape on the margination dynamics: (B) spherical particles tend to remain in the center of the flow, whereas (C) rod-shaped particles tend to marginate and drift toward the endothelial walls, binding to the wall receptors and extravasating the vessels through the gaps between endothelial cells. Reproduced with permission from E. Blanco, H. Shen, M. Ferrari, Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 33 (2015) 941 951, with permission from Nature Publishing Group, copyright 2015.

10.2.3 Charge Other surface properties, such as charge, also affect the pharmacokinetics and biodistribution of the administered NPs, mainly due to the influence on the protein adsorption and half-life circulation in the bloodstream, and also the cellular uptake and modulation of the immune response [39,47]. The protein corona formation as a consequence of the adsorption of serum proteins onto the NP surface affects the half-life of NPs in the blood circulation, altering their surface charge and masking functional groups, and, ultimately, affecting their biodistribution [61]. Compared to the highly cationic NPs, the negatively charged and neutral ones exhibit an extended circulating half-life in the bloodstream, due to the reduced adsorption of serum proteins [62]. Furthermore, positively charged NPs present higher nonspecific uptake in the majority of the cells, mainly due to their interaction with the negatively charged phospholipid head groups or protein domains on cell surfaces [47,63]. The surface charge of the NPs also plays a role in the modulation of the immune response, particularly on the activation of DCs. Yan et al. fabricated cationic liposomes that

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generated reactive oxygen species after incubation with mouse bone marrow-derived dendritic cells (BMDCs), leading to the activation of ERK and p38, and consequent production of cytokines, chemokines, and costimulatory molecules CD80 and CD86 [64]. In another study, Fromen et al. studied the effect of the NP surface charge, while maintaining a constant size and shape of the prepared NPs, as well as the antigen loading (OVA) [65]. Here, the BMDCs treated with cationic OVA-conjugated NPs induced an increased expression of MHC class II and costimulatory molecules CD80/CD86 on the DC surface, leading to a stronger T-cell receptor engagement and more vigorous T-cell activation. However, the mRNA levels of H2-Aa (MHC class II encoding) did not change, suggesting that upregulation of the MHC class II on the DC surface occurs posttranscriptionally. In addition, the cationic OVA-conjugated NPs also significantly increased the expression of IL-6 and IL-12 mRNA and protein secretion by DCs compared with negatively charged OVA-conjugated NPs. Overall, the NP charge seems to be a critical parameter when designing pulmonary therapeutics.

10.2.4 Flexibility/Elastic Modulus The NP flexibility or elasticity, given by their elastic or young modulus, is another important parameter that alters the blood circulation time, endocytosis, and phagocytosis [66]. Furthermore, the elasticity of the particulate systems can also modify the cellular uptake and tissue targeting due to their ability to bind cell surface receptors and squeeze through pores. Generally, the softer particles, that is, low-flexible particles, are more internalized by different cell lines compared with the stiffer or high-flexible particles [67 69]. For example, Key et al. synthesized soft- and rigid-discoidal polymeric nanoconstructs (DNPs), presenting the same geometry and moderate negative surface charge, but exhibiting different mechanical stiffness (B1.3 and 15 kPa, respectively) [70]. After injection in mice bearing brain or skin tumors, the soft DNPs presented an accumulation of approximately 20% of the injected dose per gram tumor and c.24 h half-time circulation in the bloodstream, due to diminished sequestration by the mononuclear phagocyte system. In addition, the mechanism for cellular uptake can also be influenced by the elasticity of the particles: soft NPs were found to be internalized by micropinocytosis, whereas stiff NPs were taken up by a clathrinmediated mechanism [71].

10.2.5 Surface Chemistry and Roughness Intrinsic nanomaterial characteristics, such as hydrophobicity and roughness, influence nonspecific attractive forces that promote cellular contact and NP uptake [63,72]. Here, the film tension model proposes that NPs more hydrophobic than the surface membrane are more willingly taken up than the less hydrophobic ones [63,73]. Moreover, the surface chemistry, namely certain hydrophobic moieties, can be recognized as DAMPs and PAMPs, and consequently, influence the activation of the immune system, as a consequence of the immune adjuvant effect of the NPs’ hydrophobicity [74]. Moyano et al. functionalized gold NPs with different degrees of hydrophobicity, and measured the effect of the hydrophobicity on the

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immune response through the cytokine expression levels, after exposing splenocytes to the NPs [75]. They showed a direct correlation between the hydrophobicity of NPs and cytokine expression, where the increase in the hydrophobicity of NPs induced an increased immune activity both in vitro and in vivo (Fig. 10 5). Similarly, Shahbazi et al. demonstrated the effect of porous silicon (PSi) NPs with different surface chemistries after incubation with human monocyte-derived dendritic cells (MDDCs) and lymphocytes [76]. Compared to other surface functionalization of PSi NPs, the thermally oxidized PSi (TOPSi) and thermally hydrocarbonized PSi (THCPSi) NPs were found to significantly stimulate the immunoactivation by enhancing the expression of surface costimulatory molecules on the MDDCs (e.g., CD80, CD83, CD86, and HLA-DR), and production of several interleukins (IL-1β, IL-4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α), as well as inducing T-cell proliferation. Generally, NPs presenting higher C H structures on the surface showed a higher immunostimulatory effect when compared to NPs exhibiting higher content of nitrogen and oxygen. Overall, these results showed the significant potential of the hydrophobic moieties on the NPs in modulating the immune responses toward NPs. The surface roughness of NPs, characterized by local protrusions or depressions with a smaller area or ratio than that of the NPs, dictates the magnitude of the NP cell interactions. Simulations of the interactions of synthetic membranes with NPs indicated that the

FIGURE 10–5 (A) Chemical structure of the prepared gold NPs composed by a passivating tetra(ethylene glycol) spacer (green area) to remove possible background effects from the hydrophobic NP core (gray zone). Then, different functional groups (R, blue) were added to the ligand termini to control the surface hydrophobicity, and the respective log P was calculated according to the hydrophobic values of the headgroups. (B) TNF-α (a representative proinflammatory cytokine) in vitro gene expression and (C) IL-10 (a representative antiinflammatory cytokine) in vivo gene expression, as a function of the calculated AuNP headgroup log P. Adapted with permission from D.F. Moyano, M. Goldsmith, D.J. Solfiell, D. Landesman-Milo, O.R. Miranda, D. Peer, M. Vincent, V.M. Rotello, Nanoparticle hydrophobicity dictates immune response. J. Am. Chem. Soc. 134 (2012) 3965 3967, with permission from the American Chemical Society, © 2012.

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roughness of the surface significantly reduces repulsive interactions, such as electrostatic and hydrophilic, leading to increased adhesion to the cells, and consequently, easier NP internalization by the cells [63,77].

10.2.6 Adjuvants Molecular adjuvants, in combination with vaccine antigens, are used in cancer vaccines to boost the immune system to kill tumor cells, by stimulating the innate and adaptive immune response [78]. Several immunostimulatory adjuvants have been included in the formulation of nanovaccines, including TLR ligands, C-type lectin receptors (CLRs), and stimulator of interferon genes (STING) [78,79]. Table 10 2 presents an overview of the different classes of molecular adjuvants and their influence on the immune responses. The most important class of adjuvants used for the development of cancer vaccines is the several TLR agonists [97]. After recognizing PAMPs, TLRs induce a signaling cascade that leads to the activation of transcription factors, such as nuclear factor-κB (NF-κB) in APCs, and ultimately, upregulates the costimulatory molecules (e.g., CD80 and CD86), induces the secretion of different cytokines (TNF-α, TGF-β, IL-12, and IL-1), and increases the surface expression of MHC classes I and II [98,99]. Consequently, immunostimulatory molecules that activate TLRs play a critical role in the formulation of particulate systems for enhancing the immune responses. C-type lectin receptors are another class of PRRs that can be used as a target for their corresponding agonists. Similarly to TLRs, CLR signaling is implicated in the initiation of innate immune responses and stimulation of secretion of different cytokines and IFNs. Furthermore, they contribute to the endocytosis and antigen presentation by APCs to induce adaptive immune responses, and they may also stimulate the activation of acquired immunity [100]. For example, DCs express CLRs, such as DEC-205 and DC-specific ICAM-3grabbing nonintegrin (DC-SIGN), which work as endocytic receptors to take up the antigens for degradation and processing. After loading of the antigens onto MHC class II, they are presented to CD41 T cells, and when antigens are presented onto MHC class II, they stimulate the CD81 T-cell response [100 102]. Another approach developed to boost the immune system against tumors is to use stimulator of interferon genes (STING) agonists [103]. Several cyclic dinucleotides (CDNs), such as cyclic guanosine monophosphate (cGAMP), cyclic diguanylate, and cyclic diadenylate, have been explored as STING agonists to boost the antitumor immune response when used as a vaccine adjuvant or an immunotherapeutic agent [79]. The STING pathway is activated in the presence of cytosolic CDNs, which are detected by the sensor cyclic-GMP-AMP synthase and generate cyclic GMP-AMP. The binding of cyclic GMP-AMP to STING activates the STING pathway within tumor-resident DCs, leading to the production of type I IFN, linked to tumor T-cell infiltration, and consequent adaptive immune responses against tumors [96]. The intratumoral injection of STING agonists has shown therapeutic effects in several mouse tumor models, such as melanoma, breast, prostate, colon, and fibrosarcoma [103].

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Table 10–2 Summary of the Main Immunostimulatory Adjuvants and Their Effect in the Immune Response Molecular Adjuvant

Target

Immune Response

References

Pam3Cys

TLR-2

[80 83]

Polyinosinic-polycytidylic acid (poly I:C) MPLALPS

TLR-3 TLR-4

Imiquimod resiquimod (R848)

TLR-7/8

Cytosine phosphate guanine oligodeoxynucleotides (CPG ODN)

TLR-9

Induce DC maturation, resulting in the upregulation of costimulatory signals and Ag-presenting molecules (e.g., MHC class II, CD80, CD83, IFN-γ, IL-12) Stimulates the maturation and activation of B cells that leads to an increased production of Ag-specific IgG and IgM Abs Activates antigen-specific antibody production, CTL and Th1 type immune responses Induces functional CTLs specific to tumor-associated antigens Boosts the maturation of DCs and activation of antigenspecific T-cell immune responses Increases type I IFN and IFN-γ at the peptide vaccination site after DC activation Enhances antigen-specific T-cell responses Increases the antitumor response sufficiently to mediate regression of in-transit melanoma metastasis Improves the function of APCs and antigen presentation Boosts the generation of humoral and cellular vaccinespecific immune responses

TLR Ligands

[84,85] [86 89]

[90,91]

[92,93]

CLRs β-Glucan

Dectin-1 Induces a systemic tumor-antigen specific T-cell response after DC activation Increases the infiltration of the activated T cells into the tumor Decreases immunosuppressive cells in tumor-bearing mice Augments the therapeutic efficacy mediated by antitumor mAbs

[94,95]

STINGs

[96]

STING Cyclic di-GMP

Induces a STING-dependent antitumor activity, as a consequence of an increased activation of DCs and tumor antigen-specific CD81 T cells

CLRs, C-type lectin receptors; CTL, cytotoxic T lymphocyte; LPS, lipopolysaccharides; MHC, major histocompatibility complex; MPLA, monophosphoryl lipid A; STING, stimulator of interferon genes; TLR, Toll-like receptor.

10.2.7 Position of the Antigens In addition to the influence of the physicochemical properties of NPs on their adjuvant effect, another key factor that affects the kinetics of antigen exposure to the immune system, and the subsequent antigen-specific immune response, is the position of the antigen on the NP formulation. For example, Liu et al. prepared three different formulations, where the

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FIGURE 10–6 (A) Confocal microscopy of BMCDs after 16 h incubation with OVA-adsorbed NPs (out), OVAencapsulated NPs (in) OVA-encapsulated/adsorbed NPs (both) and free OVA. FITC-OVA (green), lysosomes stained with Lyso-Tracker (red), and the merged images (yellow). (B, C) Production of antigen-specific IgG titers in the sera of C57BL/6 mice, at the indicated time points. (D) Ratio of IgG2a/IgG1. Data are expressed as the mean 6 SD (n 5 6) ( P , .05). Adapted with permission from L. Liu, P. Ma, H. Wang, C. Zhang, H. Sun, C. Wang, et al., Immune responses to vaccines delivered by encapsulation into and/or adsorption onto cationic lipid-PLGA hybrid nanoparticles, J. Controlled Release 225 (2016) 230 239, with Elsevier B.V., © 2016.

antigen was adsorbed and/or encapsulated into cationic lipid-poly(lactide-co-glycolide) acid (PLGA) hybrid NPs [104]. Their results showed that lysosomal escape and cross-presentation of the antigen from DCs were more efficient when OVA was encapsulated or both encapsulated/adsorbed into NPs (Fig. 10 6A), and also an increased in vivo antigen-specific immune response (Fig. 10 6B D), compared with the mice immunized with OVA-adsorbed NPs and free OVA. Zhang et al. also verified that the adsorbed/encapsulated-antigen PLGA NPs induced more powerful antigen-specific immune responses than the formulations where the antigen was either adsorbed or entrapped in the NPs [105]. Consequently, mice immunized with an adsorbed/encapsulated-antigen PLGA NPs formulation presented increased cytokine secretion by splenocytes, improved stimulation of antigen-specific IgG antibodies with high avidity, as well as improved generation of memory T cells. This effect can be ascribed to the antigendepot effect at the injection site and an adequate initial antigen exposure and long-term antigen persistence. Furthermore, the efficient activation of DCs and follicular helper T-cell differentiation in draining lymph nodes also contribute to the enhanced immune response. Overall, these results indicated that the location of the antigen in the NP formulation is important to modulate the immune response of the antigens delivered by the NPs, while designing the nanovaccines.

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10.3 In Vitro Assessment An in vitro preliminary assessment allows the screening of multiple formulations with identification of the most promising one, in a process aimed at reducing the number of animals used in the development. However, the reproducibility of the assays and their translatability in vivo need to be verified with different sets of NPs [106], together with the investigation of the possible interferences between the nanosystem and the assay components to avoid false negatives or false positives [107]. The first step in the development of a novel nanoimmunotherapeutic (and of every NP) is the assessment of its cytocompatibility over murine and human cell lines, followed by evaluation of the impurities derived from the preparation process [108]. Such impurities may affect the end result of the immunological screening [109]. The parameters described in Section 10.2, all have an effect on the compatibility of the NPs. For example, the surface chemistry of PSi NPs greatly affects their interactions with different cells and their immunomediated toxicity, as evaluated by diverse viability assays, to investigate the different mechanisms of toxicity [110]. The different surface chemistry results also in different interactions with the cells of the immune system, as shown in Fig. 10 7A. As for the size of the NPs, usually smaller particles are more toxic than larger ones, because they present an intrinsic higher surface area, thereby they are more reactive, with the possibility of creating reactive oxygen or nitrogen species [111]. The possible toxicity mechanisms are summarized in Fig. 10 7B. Then, the immunogenicity of particles and their suitability as intrinsic vaccine adjuvants is evaluated into murine and/or human immune cells.

10.3.1 Murine Cells The in vitro murine models commonly investigated are based either on cell lines or on ex vivo derived immune cells. Raw 264.7 macrophages are routinely employed to evaluate the uptake of different NPs with cells of the reticuloendothelial system [112,113]. Another cell line routinely employed is JAWS II, constituted by immature bone marrow-derived monocytes [114]. These cells can be pulsed in vitro with the nanovaccine formulation to evaluate the particles’ immunological properties [115]. Innovative vaccine nanodiscs, constituted of synthetic high-density lipoproteins, represent a stable and versatile vaccine formulation, which allows the delivery of multiple antigens and adjuvants [116]. The uptake and intracellular pathway of the formulations were studied by confocal microscopy, highlighting the enhanced efficacy of the nanodiscs in internalizing the antigen. Qiu et al. developed simple yet effective nanocomplexes of antigen and adjuvant, coated them with modified polyphenol to facilitate the endosomal escape, and evaluated their uptake on JAWS II cells, by means of confocal microscopy and flow cytometry [117]. The cell lines can be employed also to evaluate the proinflammatory properties of the particles, by studying the cytokine profile in the medium after incubation with the particles by quantification with enzymelinked immunosorbent assay [118].

FIGURE 10–7 (A) Interaction of PSi NPs characterized by different surface chemistry together with murine immune cells (Raji, Jurkat, U937, and Raw 264.7). Thermally oxidized PSi (TOPSi), thermally carbonized PSi (TCPSi), (3-aminopropyl) triethoxysilane TCPSi (APTSTCPSi), thermally hydrocarbonized PSi (THCPSi), and undecylenic acid-modified THCPSi (UnTHCPSi) were incubated with the cells before assessing the damage to cell morphology and membrane by scanning electron microscopy. (B) Proposed mechanisms of NP-derived toxicity on cells. (A) Reproduced with permission from M.-A. Shahbazi, M. Hamidi, E.M. Mäkilä, H. Zhang, P.V. Almeida, M. Kaasalainen, et al., The mechanisms of surface chemistry effects of mesoporous silicon nanoparticles on immunotoxicity and biocompatibility, Biomaterials, 34 (2013) 7776 7789, with Elsevier B.V., © 2013. (B) Reproduced with permission from L. Shang, K. Nienhaus, G.U. Nienhaus, Engineered nanoparticles interacting with cells: size matters, J. Nanobiotechnol. 12 (2014) 5, copyright.

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The most commonly used murine-derived cells for immunological assays are BMDC, primary cells derived from the bone marrow (mainly from tibia and femurs) and differentiated in vitro to dendritic cells [119]. As for the development of cancer vaccines, alginate NPs, loaded with a model antigen (OVA) and modified with mannose to enhance the uptake, enabled the endosomal escape of the antigen, its presentation on MHC together, with an increase in the activation profile of the BMDC, as measured by the augmented expression of costimulatory signals and secretion of cytokines [120]. Furthermore, systems constituted by a physical mixture of antigen together with polymeric NPs can induce activation of DCs thorough activation of the STING pathway, with subsequent priming of T cells and antitumoral efficacy in different tumor models [121]. In autoimmune diseases, NPs are investigated for the delivery of tolerogenic molecules together with the antigen to activated DCs, as described by Yeste et al., where they proved how the formulation of tolerogenic small molecules and antigen on gold NPs could inhibit BMDC activation through the expression of the Socs2 pathway [122]. In this study NPs were incubated with BMDC, then the cells were analyzed with arrays to evaluate the genetic expression of several activation pathways. In addition, BDMC can be pulsed with NPs before being incubated with OT-II T cells to evaluate the activation of the T cells or, as investigated by Pearson et al., the induction of antigen-specific regulatory T cells [123].

10.3.2 Human Cells Human immune cells are often used as proof of concept for the future translatability of the nanosystem or to evaluate particles’ immunotoxicity in an experimental setting closer to the real world [124]. Both immortalized and primary cells have a role in these studies. For example, KG-1 macrophages and B cells with DC morphology were incubated with a multistage nanovaccine for cancer immunotherapy to evaluate the potential of the vaccine to induce an immune response, quantified by the expression of costimulatory signals (CD80 and 86) and by the secretion of cytokines (IFN-γ, IL-2, and IL-4) [125]. Other models widely employed are monocytederived DCs (MODCs) or peripheral blood monocytes (PBMCs). PBMCs represent the fraction of immune cells isolated by buffy coat and purified with different procedures (e.g., Ficoll’s reagent) [126,127]. MODCs are derived from the isolation of pure monocytes (either by plastic adhesion or by magnetic separation of CD141 cells), before being cultured with medium supplemented with granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 [128]. MODCs served as cell model for the evaluation of the surface-dependent immunostimulatory properties of PSi NPs, as reported by Shahbazi et al., where MODCs incubated either with the hydrophobic THCPSi or with the hydrophilic and rapidly degrading TOPSi result into high activation (expression levels of costimulatory signals and secretion of cytokines), while the hydrophilic and slowly degrading TCPSi do not activate the cells (Fig. 10 8A) [76]. Innovative research lines focus on the optimization of NPs for the codelivery of immunostimulants (like GM-CSF) and targeted chemotherapeutics able to induce the immunogenic death of the cancer cells with minimal damage to immune ones. The incubation of MODCs with medium derived from the incubation of cancer cells with such nanosystems resulted in

FIGURE 10–8 (A) Surface-dependent immunogenicity of PSi particles on MODCs evaluated by the increase in the expression of costimulatory signals (CD80 and 86). Thermally oxidized PSi (TOPSi), thermally carbonized PSi (TCPSi), (3-aminopropyl)triethoxysilane TCPSi (APTSTCPSi), thermally hydrocarbonized PSi (THCPSi), undecylenic acid-modified THCPSi (UnTHCPSi), polyethylenimine-modified UnTHCPSi (UnP), and poly(methyl vinyl ether-alt-maleic acid)-modified APTSTCPSi (APM) were incubated with immature MODCs, before assessing their immunological properties by FACS. (B) Immunological analysis of nanoparticles codelivering immunostimulant (GM-CSF) and chemotherapeutic drug (Nutlin3a). (I, II) The immunostimulant properties of the bare (B) and loaded (CL) formulation were compared to those of the single drugs (N and G, or the combination of drugs in solution NG). The expression of CD83 and 86 was evaluated by FACS. (III, IV) Proliferation of T cells induced by the incubation of the drugs or the formulation with immature MODC, measured by dilution of CellTrace in FACS. MODCs, Monocyte-derived DCs. (A) Reproduced with permission from M.-A. Shahbazi, T.D. Fernández, E.M. Mäkilä, X. Le Guével, C. Mayorga, M.H. Kaasalainen, et al., Surface chemistry dependent immunostimulative potential of porous silicon nanoplatforms. Biomaterials, 35 (2014) 9224 9235, with Elsevier B.V., © 2014. (B) Reproduced with permission from T. Bauleth-Ramos, M.-A. Shahbazi, D. Liu, F. Fontana, A. Correia, P. Figueiredo, et al., Nutlin-3a and cytokine co-loaded spermine-modified acetalated dextran nanoparticles for cancer chemo-immunotherapy. Adv. Funct. Mater. 27 (2017) 1703303, with Wiley-VCH Verlag GmbH & Co., © 2017.

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increased activation of the dendritic cells, together with the priming of naïve T cells and stimulation of their proliferation, as shown in Fig. 10 8B [129]. In summary, we have presented the most common cellular models for evaluation of the cytocompatibility, cell association and uptake, and immunological properties of the particles.

10.4 In Vivo Models and Efficacy The complex interactions between nanomaterials and the cells constituting the immune system require careful planning of the most suitable animal models [130]. Moreover, statistics show that up to 80% of the new treatments proposed fail in clinical trials, despite positive results in preclinical animal studies [131].

10.4.1 Immunostimulation—Cancer There are different types of cancer animal models. These include ectopic xenografts, which are induced by injection, in different sites, of human or murine tumor cell lines into immunocompetent or -deficient animals; orthotopic models established by injecting murine cell lines in their originator tissue (e.g., breast cancer cell lines injected in mammary fat pads); germ-line transgenic, conditional transgenic models (GEMs), developed by controlling the expression of oncosuppressors or oncodrivers in a timely manner in different tissues due to genetic engineering; primary human tumor grafts, established by implanting patient-derived tumors into immune-deficient animals; and carcinogenic induced models, derived from the exposure of the animals to know carcinogenics [132]. The most common models investigated in the preclinical development of therapeutic vaccines for cancer immunotherapy are reported in Table 10 3. Usually, the studies reported focus on melanoma models, given their immunogenic characteristics. Usually, nanovaccines are evaluated for prophylactic and for therapeutic activity. In the prophylactic set, mice are immunized with the NPs before injection of the tumor cells. The aim of the study then is to evaluate whether the formulation can prevent the attachment and growth of the tumor. For example, Yang et al. investigated the prophylactic efficacy of a nanovaccine composed of PLGA NPs coated with cell membrane derived from cancer cells and modified to present mannose molecules, to facilitate uptake by APCs [140]. A preventive triple vaccination enables control over the cancer growth up to 15 days, together with priming of antitumor immune response. The prophylactic activity of similar systems (cancer cell membranes) can improve by codelivery together with adjuvant molecules (CpG oligonucleotide particles coated with the cell membrane), leading to extended control (over 30 days) on the tumor growth in B16. F10 model after three vaccinations [134].

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Table 10–3 Murine Cancer Model Employed in Assessment of the Efficacy of Nanovaccines Model

Characteristics

B16. OVA Ectopic murine melanoma model B16. OVA cells injected subcutaneously B16. F10

LLC cells

MC38

Ectopic murine melanoma model B16. F10 cells injected subcutaneously Ectopic lung cancer model LLC cells injected subcutaneously Ectopic lung metastases of colon adenocarcinoma Intravenous injection

Advantages

Disadvantages

References

Highly immunogenic

Exogenous antigen, more immunogenic Less aggressive tumor

[116,121,133]

Known antigen (OVA) Suitable to analyze the immunological changes in the tumor microenvironment and in the lymphoid organs Model more clinically related than B16. OVA Highly aggressive Lower time to establish Possibility to assess antigens different from an immune response OVA Possibility to evaluate the effect of nanoparticles with different antigens Possibility to assess the effect on the lung metastases Poorly immunogenic

[134 136]

Not yet commonly used in nanovaccine preclinical studies

[137,138]

Poorly immunogenic

[139]

Possibility to evaluate neoantigens

LLC, Lewis lung cancer.

Overall, the in vivo tumor models provide the best representation of the tumor microenvironment and the physiological responses to the in situ developed disease. Consequently, the efficacy of the tumor model can be given by measuring tumor growth after administration of the nanovaccines, as well as by evaluating the immune cells’ population at the tumor microenvironment and their immune response against the tumor tissue, including the expression of costimulatory molecules (e.g., CD80 and CD86) and production of proinflammatory cytokines.

10.4.2 Immunomodulation—Rheumatoid Arthritis, Experimental Autoimmune Encephalomyelitis, Diabetes The study of NPs as therapeutics in autoimmune diseases relies on different animal models that resemble the symptomatology of the human disease. Usually, the in vivo studies are performed over models of rheumatoid arthritis (RA), multiple sclerosis (experimental autoimmune encephalomyelitis, EAE), and diabetes [42]. The models of RA are established by active or passive immunization or by genetic modification leading to spontaneous insurgence of the disease: active immunization is achieved by subcutaneous injection of exogenous collagen (from a different animal species) together with a strong adjuvant (like Freund’s) into the mouse, followed by a rechallenge 3 weeks after the

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first injection [141]. Passive immunization is achieved by administration of anticollagen antibodies or antibodies derived from K/BxN mice (transgenic model that spontaneously develop the pathology). Finally, the transgenic models spontaneously developing the disease are K/BxN or TNFΔARE, which features deletions in the transcription of TNF-α control elements, thereby prompting elevated expression of TNF-α [141]. The nanosystems assessed for the therapy of RA are employed as drug-delivery systems for the disease-modifying drugs (like methotrexate) or immunosuppressant (tacrolimus), often relying on either the passive targeting of nanoparticles to macrophages or by active targeting by folate receptor [142 144]. An interesting approach focuses on biomineral hyaluronan (an hydrophobic core of cholanic acid, calcium phosphate, and a hydrophilic coating of hyaluronan) particles coloaded with methotrexate and doxorubicin, and PEGylated, as shown in Fig. 10 9A. The authors showed preferential distribution of the particles to the inflamed tissue and to macrophages, skewing monocytes and liver, with improvement in the scores of a collagen-induced animal model [145]. As for biohybrid systems, an innovative system constituted by immunoneutral PSi NPs coated with cell membrane isolated by macrophages was proposed as a drug-delivery system and immunomodulatory vaccination [146]. The murine model of multiple sclerosis, EAE, is established by immunization with myeline antigens or tissue fragments from the central nervous system together with strong adjuvants (complete Freund’s adjuvant), where this model is highly translatable in different animal species, from rodents to monkeys [147]. As for the applications of nanotechnology for the treatment of multiple sclerosis, the areas investigated are the development of innovative, precise nanoimaging diagnostics, and the targeted delivery of antiinflammatory drugs (e.g., glucocorticoids) [148,149]. Diabetes animal models in immunotherapy research are either spontaneously developing the disease (nonobese diabetic mice and biobreeding diabetes-prone rats), or are treated with compounds destroying β-cells (e.g., streptozotocin and viral infection) in experimentally induced diabetes models [150]. Nanotechnology has been investigated to enable the oral delivery of insulin [151,152]. Recently, the application of nanotechnology in the field of immunotherapy for autoimmune diseases moved from drug-delivery systems to the delivery of antigens to induce tolerogenicity [153]. Nanovaccines constituted by different particles have been assessed so far, where iron oxide NPs carrying MHC II already loaded with disease-relevant peptides showed induction of a tolerogenic response (with the priming of Treg cells) effectively treating autoimmune diseases (diabetes, RA, and EAE) and not only their symptoms [154]. Other materials investigated are polymeric NPs codelivering disease-specific antigen together with immunosuppressant (rapamycin) or tolerogenic nanovaccines delivering antigens (Fig. 10 9B) [155,156]. Overall, this section has emphasized the importance of the choice of a proper animal model for evaluating the efficacy of nanosystems for immunomodulation.

FIGURE 10–9 (A) Hypothesized mode of action of drug-loaded mineralized pegylated particles in RA: upon cellular uptake, the particles localize in the endosomes. The presence of calcium phosphate in the particles facilitates the disruption of the endosome, leading to the intracellular release of metothrexate and doxorubicin. The presence of an external layer of hyaluronic acid enables targeting to inflamed macrophages in the disease tissue. (B) Tolerogenic poly(D,L-lactide-co-glycolide) NPs carrying disease-specific antigen. The efficacy of the disease-relevant antigen is shown in comparison to OVA antigen, in the effect on the mean clinical score in an EAE model and on the presence of active lesions. RA, Rheumatoid arthritis. (A) Reproduced with permission from M.M. Alam, H.S. Han, S. Sung, J.H. Kang, K.H. Sa, H. Al Faruque, et al., Endogenous inspired biomineral-installed hyaluronan nanoparticles as pH-responsive carrier of methotrexate for rheumatoid arthritis, J. Controlled Release 252 (2017) 62 72, with Elsevier B.V., © 2017. (B) Reproduced with permission from Z. Hunter, D.P. McCarthy, W.T. Yap, C.T. Harp, D.R. Getts, L.D. Shea, et al., A biodegradable nanoparticle platform for the induction of antigen-specific immune tolerance for treatment of autoimmune disease, ACS Nano 8 (2014) 2148 2160, with American Chemical Society, © 2014.

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10.5 Conclusions Research into NPs has shifted toward applications in immunotherapy. Nanosized systems represent the optimal choice to carry adjuvant for vaccines, whether for infective diseases, cancer, or inducing of tolerogenicity in autoimmune diseases. The physical properties of particles (size, surface charge, surface morphology, elasticity, aspect ratio, surface chemistry, and hydrophobicity) have an influence on the interactions between the nanosystem and the cells, that is, the circulation time, extravasation, distribution in tissues, and interaction with and uptake by cells. A rational design of nanovaccines cannot preclude from an analysis of such parameters to fine tune the formulation according to the desired characteristics. The following stage in the development of the nanosystems relies on established in vitro models to evaluate the cytocompatibility, interaction, uptake mechanisms, and immunological profile: particular importance should be placed on the choice of proper assays, evaluating possible interactions between the particles and the assays. Finally, the interactions between NPs and the immune system have to be investigated in complex systems, preclinical animal models, where the vaccine will encounter a complete immune system, and not only isolated components. The abundance of models available requires immunological competence to properly identify the optimal model to retrieve the most information. Along with the development of new materials and preparation methods of NPs, the delivery of antigens and immunomodulatory molecules to the APCs seems to modulate the immune response and improve the clinical outcome. Overall, we envision in the near future the development of multiple NPs for immunotherapy, based on a rational design of each system.

Acknowledgments Financial support from the Sigrid Jusélius Foundation (decision no. 4704580), the HiLIFE Research Funds, and the European Research Council under the European Union’s Seventh Framework Programme (FP/20072013, grant no. 310892) are greatly acknowledged.

References [1] D.J. Irvine, M.C. Hanson, K. Rakhra, T. Tokatlian, Synthetic nanoparticles for vaccines and immunotherapy, Chem. Rev. 115 (19) (2015) 11109 11146. [2] K.M. Murphy, P. Travers, M. Walport, Janeway’s Immunobiology (Immunobiology: The Immune System (Janeway)), Garland Science, 2007. [3] W.K. Decker, R.F. da Silva, M.H. Sanabria, L.S. Angelo, F. Guimaraes, B.M. Burt, et al., Cancer immunotherapy: historical perspective of a clinical revolution and emerging preclinical animal models, Front. Immunol. 8 (2017) 829. [4] A. Iwasaki, R. Medzhitov, Control of adaptive immunity by the innate immune system, Nat. Immunol. 16 (4) (2015) 343 353.

Chapter 10 • Advanced Nanovaccines for Immunotherapy Applications

253

[5] J. Neefjes, M.L. Jongsma, P. Paul, O. Bakke, Towards a systems understanding of MHC class I and MHC class II antigen presentation, Nat. Rev. Immunol. 11 (12) (2011) 823 836. [6] P.A. Roche, K. Furuta, The ins and outs of MHC class II-mediated antigen processing and presentation, Nat. Rev. Immunol. 15 (4) (2015) 203 216. [7] S.W. Brubaker, K.S. Bonham, I. Zanoni, J.C. Kagan, Innate immune pattern recognition: a cell biological perspective, Annu. Rev. Immunol. 33 (2015) 257 290. [8] A. Liston, S.L. Masters, Homeostasis-altering molecular processes as mechanisms of inflammasome activation, Nat. Rev. Immunol. 17 (3) (2017) 208. [9] L. Galluzzi, A. Buque, O. Kepp, L. Zitvogel, G. Kroemer, Immunogenic cell death in cancer and infectious disease, Nat. Rev. Immunol. 17 (2) (2017) 97 111. [10] B. Kyewski, L. Klein, A central role for central tolerance, Annu. Rev. Immunol. 24 (2006) 571 606. [11] N. Coronel-Restrepo, I. Posso-Osorio, J. Naranjo-Escobar, G.J. Tobón, Autoimmune diseases and their relation with immunological, neurological and endocrinological axes, Autoimmun. Rev. 16 (7) (2017) 684 692. [12] A.N. Theofilopoulos, D.H. Kono, R. Baccala, The multiple pathways to autoimmunity, Nat. Immunol. 18 (7) (2017) 716 724. [13] M.L. Mallory, L.C. Lindesmith, R.S. Baric, Vaccination-induced herd immunity: successes and challenges, J. Allergy Clin. Immunol. 142 (1) (2018) 64 66. [14] Z.A. Bornholdt, S.B. Bradfute, Ebola virus vaccination and the longevity of total versus neutralising antibody response-is it enough?, Lancet Infect. Dis. 18 (7) (2018) 699 700. [15] B.F. Haynes, J.R. Mascola, The quest for an antibody-based HIV vaccine, Immunol. Rev. 275 (1) (2017) 5 10. [16] L. Lambricht, C. Peres, H. Florindo, V. Préat, G. Vandermeulen, Polymer-based nanoparticles as modern vaccine delivery systems, Micro and Nanotechnology in Vaccine Development, Elsevier, 2017, pp. 185 203. [17] R. Angioli, S. Lopez, A. Aloisi, C. Terranova, C. De Cicco, G. Scaletta, et al., Ten years of HPV vaccines: state of art and controversies, Crit. Rev. Oncol. Hematol. 102 (2016) 65 72. [18] I. Melero, G. Gaudernack, W. Gerritsen, C. Huber, G. Parmiani, S. Scholl, et al., Therapeutic vaccines for cancer: an overview of clinical trials, Nat. Rev. Clin. Oncol. 11 (9) (2014) 509 524. [19] M.M. Zhan, X.Q. Hu, X.X. Liu, B.F. Ruan, J. Xu, C. Liao, From monoclonal antibodies to small molecules: the development of inhibitors targeting the PD-1/PD-L1 pathway, Drug Discov. Today 21 (6) (2016) 1027 1036. [20] J.L. Adams, J. Smothers, R. Srinivasan, A. Hoos, Big opportunities for small molecules in immunooncology, Nat. Rev. Drug Discov. 14 (9) (2015) 603 622. [21] M.T. Osterholm, N.S. Kelley, A. Sommer, E.A. Belongia, Efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis, Lancet Infect. Dis. 12 (1) (2012) 36 44. [22] P.B. Bach, Gardasil: from bench, to bedside, to blunder, Lancet 375 (9719) (2010) 963 964. [23] J. Paavonen, P. Naud, J. Salmeron, C.M. Wheeler, S.N. Chow, D. Apter, et al., Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women, Lancet 374 (9686) (2009) 301 314. [24] P.W. Kantoff, C.S. Higano, N.D. Shore, E.R. Berger, E.J. Small, D.F. Penson, et al., Investigators, Sipuleucel-T immunotherapy for castration-resistant prostate cancer, N. Engl. J. Med. 363 (5) (2010) 411 422. [25] S.A. Rosenberg, J.C. Yang, N.P. Restifo, Cancer immunotherapy: moving beyond current vaccines, Nat. Med. 10 (9) (2004) 909 915.

254

THERANOSTIC BIONANOMATERIALS

[26] O. Tuohy, L. Costelloe, G. Hill-Cawthorne, I. Bjornson, K. Harding, N. Robertson, et al., Alemtuzumab treatment of multiple sclerosis: long-term safety and efficacy, J. Neurol. Neurosurg. Psychiatry 86 (2) (2015) 208 215. [27] P. Vasanthi, G. Nalini, G. Rajasekhar, Role of tumor necrosis factor-alpha in rheumatoid arthritis: a review, APLAR J. Rheumatol. 10 (4) (2007) 270 274. [28] J.D. Isaacs, M. Cutolo, E.C. Keystone, W. Park, J. Braun, Biosimilars in immune-mediated inflammatory diseases: initial lessons from the first approved biosimilar anti-tumour necrosis factor monoclonal antibody, J. Intern. Med. 279 (1) (2016) 41 59. [29] D.M. Pardoll, The blockade of immune checkpoints in cancer immunotherapy, Nat. Rev. Cancer 12 (4) (2012) 252 264. [30] P.M. Glassman, J.P. Balthasar, Mechanistic considerations for the use of monoclonal antibodies for cancer therapy, Cancer Biol. Med. 11 (1) (2014) 20. [31] J.F. Ferreira, A.A. Ahmed Mohamed, P. Emery, Glucocorticoids and rheumatoid arthritis, Rheum. Dis. Clin. North Am. 42 (1) (2016) 33 46. vii. [32] P.M. Brown, A.G. Pratt, J.D. Isaacs, Mechanism of action of methotrexate in rheumatoid arthritis, and the search for biomarkers, Nat. Rev. Rheumatol. 12 (12) (2016) 731. [33] S.N. Sehgal, Rapamune (Sirolimus, rapamycin): an overview and mechanism of action, Therap. Drug Monit. 17 (6) (1995) 660 665. [34] K.T. Gause, A.K. Wheatley, J. Cui, Y. Yan, S.J. Kent, F. Caruso, Immunological principles guiding the rational design of particles for vaccine delivery, ACS Nano 11 (1) (2017) 54 68. [35] O.C. Farokhzad, R. Langer, Impact of nanotechnology on drug delivery, ACS Nano 3 (1) (2009) 16 20. [36] J. Shi, A.R. Votruba, O.C. Farokhzad, R. Langer, Nanotechnology in drug delivery and tissue engineering: from discovery to applications, Nano Lett. 10 (9) (2010) 3223 3230. [37] S.M. Janib, A.S. Moses, J.A. MacKay, Imaging and drug delivery using theranostic nanoparticles, Adv. Drug Deliv. Rev. 62 (11) (2010) 1052 1063. [38] H. Chen, Y. Zhao, H. Wang, G. Nie, K. Nan, Co-delivery strategies based on multifunctional nanocarriers for cancer therapy, Curr. Drug Metab. 13 (8) (2012) 1087 1096. [39] F. Fontana, P. Figueiredo, T. Bauleth-Ramos, A. Correia, H.A. Santos, Immunostimulation and immunosuppression: nanotechnology on the brink, Small Methods 2 (5) (2018) 1700347. [40] D.M. Smith, J.K. Simon, J.R. Baker Jr., Applications of nanotechnology for immunology, Nat. Rev. Immunol. 13 (8) (2013) 592 605. [41] Y. Hailemichael, Z. Dai, N. Jaffarzad, Y. Ye, M.A. Medina, X.-F. Huang, et al., Persistent antigen at vaccination sites induces tumor-specific CD81 T cell sequestration, dysfunction and deletion, Nat. Med. 19 (4) (2013) 465. [42] P. Serra, P. Santamaria, Nanoparticle-based autoimmune disease therapy, Clin. Immunol. 160 (1) (2015) 3 13. [43] Y. Krishnamachari, S.M. Geary, C.D. Lemke, A.K. Salem, Nanoparticle delivery systems in cancer vaccines, Pharm. Res. 28 (2) (2011) 215 236. [44] K. Huang, H. Ma, J. Liu, S. Huo, A. Kumar, T. Wei, et al., Size-dependent localization and penetration of ultrasmall gold nanoparticles in cancer cells, multicellular spheroids, and tumors in vivo, ACS Nano 6 (5) (2012) 4483 4493. [45] N. Hoshyar, S. Gray, H. Han, G. Bao, The effect of nanoparticle size on in vivo pharmacokinetics and cellular interaction, Nanomedicine 11 (6) (2016) 673 692. [46] H. Soo Choi, W. Liu, P. Misra, E. Tanaka, J.P. Zimmer, B. Itty Ipe, et al., Renal clearance of quantum dots, Nature Biotechnology 25 (2007) 1165.

Chapter 10 • Advanced Nanovaccines for Immunotherapy Applications

255

[47] E. Blanco, H. Shen, M. Ferrari, Principles of nanoparticle design for overcoming biological barriers to drug delivery, Nat. Biotechnol. 33 (9) (2015) 941 951. [48] M.F. Bachmann, G.T. Jennings, Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns, Nat. Rev. Immunol. 10 (11) (2010) 787 796. [49] N. Benne, J. van Duijn, J. Kuiper, W. Jiskoot, B. Slütter, Orchestrating immune responses: how size, shape and rigidity affect the immunogenicity of particulate vaccines, J. Controlled Release 234 (2016) 124 134. [50] S.T. Reddy, A.J. van der Vlies, E. Simeoni, V. Angeli, G.J. Randolph, C.P. O’Neil, et al., Exploiting lymphatic transport and complement activation in nanoparticle vaccines, Nat. Biotechnol. 25 (10) (2007) 1159 1164. [51] R. Kenneth, S. Lianjun, Cross-presentation: underlying mechanisms and role in immune surveillance, Immunol. Rev. 207 (1) (2005) 166 183. [52] A. Savina, C. Jancic, S. Hugues, P. Guermonprez, P. Vargas, I.C. Moura, et al., NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells, Cell 126 (1) (2006) 205 218. [53] E.S. Trombetta, M. Ebersold, W. Garrett, M. Pypaert, I. Mellman, Activation of lysosomal function during dendritic cell maturation, Science 299 (5611) (2003) 1400. [54] J.M. Brewer, K.G.J. Pollock, L. Tetley, D.G. Russell, Vesicle size influences the trafficking, processing, and presentation of antigens in lipid vesicles, J. Immunol. 173 (10) (2004) 6143. [55] K.K. Tran, H. Shen, The role of phagosomal pH on the size-dependent efficiency of cross-presentation by dendritic cells, Biomaterials 30 (7) (2009) 1356 1362. [56] N.P. Truong, M.R. Whittaker, C.W. Mak, T.P. Davis, The importance of nanoparticle shape in cancer drug delivery, Expert Opin. Drug Deliv. 12 (1) (2015) 129 142. [57] R. Toy, P.M. Peiris, K.B. Ghaghada, E. Karathanasis, Shaping cancer nanomedicine: the effect of particle shape on the in vivo journey of nanoparticles, Nanomedicine (London, England) 9 (1) (2014) 121 134. [58] X. Huang, X. Teng, D. Chen, F. Tang, J. He, The effect of the shape of mesoporous silica nanoparticles on cellular uptake and cell function, Biomaterials 31 (3) (2010) 438 448. [59] J. Tan, S. Shah, A. Thomas, H.D. Ou-Yang, Y. Liu, The influence of size, shape and vessel geometry on nanoparticle distribution, Microfluidics Nanofluidics 14 ((1-2) (2013) 77 87. [60] S. Kumar, A.C. Anselmo, A. Banerjee, M. Zakrewsky, S. Mitragotri, Shape and size-dependent immune response to antigen-carrying nanoparticles, J. Controlled Release 220 (2015) 141 148. [61] P. Maffre, S. Brandholt, K. Nienhaus, L. Shang, W.J. Parak, G.U. Nienhaus, Effects of surface functionalization on the adsorption of human serum albumin onto nanoparticles a fluorescence correlation spectroscopy study, Beilstein J. Nanotechnol. 5 (2014) 2036 2047. [62] F. Alexis, E. Pridgen, L.K. Molnar, O.C. Farokhzad, Factors affecting the clearance and biodistribution of polymeric nanoparticles, Mol. Pharm. 5 (4) (2008) 505 515. [63] A.E. Nel, L. Mädler, D. Velegol, T. Xia, E.M.V. Hoek, P. Somasundaran, et al., Understanding biophysicochemical interactions at the nano bio interface, Nat. Mater. 8 (2009) 543. [64] W. Yan, W. Chen, L. Huang, Reactive oxygen species play a central role in the activity of cationic liposome based cancer vaccine, J. Controlled Release 130 (1) (2008) 22 28. [65] C.A. Fromen, G.R. Robbins, T.W. Shen, M.P. Kai, J.P. Ting, J.M. DeSimone, Controlled analysis of nanoparticle charge on mucosal and systemic antibody responses following pulmonary immunization, Proc. Natl. Acad. Sci. U.S.A. 112 (2) (2015) 488 493. [66] A.C. Anselmo, M. Zhang, S. Kumar, D.R. Vogus, S. Menegatti, M.E. Helgeson, et al., Elasticity of nanoparticles influences their blood circulation, phagocytosis, endocytosis, and targeting, ACS Nano 9 (3) (2015) 3169 3177.

256

THERANOSTIC BIONANOMATERIALS

[67] W. Liu, X. Zhou, Z. Mao, D. Yu, B. Wang, C. Gao, Uptake of hydrogel particles with different stiffness and its influence on HepG2 cell functions, Soft Matter 8 (35) (2012) 9235 9245. [68] L. Zhang, Q. Feng, J. Wang, S. Zhang, B. Ding, Y. Wei, et al., Microfluidic synthesis of hybrid nanoparticles with controlled lipid layers: understanding flexibility-regulated cell nanoparticle interaction, ACS Nano 9 (10) (2015) 9912 9921. [69] P. Guo, D. Liu, K. Subramanyam, B. Wang, J. Yang, J. Huang, et al., Nanoparticle elasticity directs tumor uptake, Nat. Commun. 9 (1) (2018) 130. [70] J. Key, A.L. Palange, F. Gentile, S. Aryal, C. Stigliano, D. Di Mascolo, et al., Soft discoidal polymeric nanoconstructs resist macrophage uptake and enhance vascular targeting in tumors, ACS Nano 9 (12) (2015) 11628 11641. [71] X. Banquy, F. Suarez, A. Argaw, J.-M. Rabanel, P. Grutter, J.-F. Bouchard, et al., Effect of mechanical properties of hydrogel nanoparticles on macrophage cell uptake, Soft Matter 5 (20) (2009) 3984 3991. [72] D.F. Moyano, Y. Liu, D. Peer, V.M. Rotello, Modulation of immune response using engineered nanoparticle surfaces, Small (Weinheim an der Bergstrasse, Germany) 12 (1) (2016) 76 82. [73] H. Chen, R. Langer, D.A. Edwards, A. Film Tension, Theory of phagocytosis, J. Colloid Interface Sci. 190 (1) (1997) 118 133. [74] S.Y. Seong, P. Matzinger, Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses, Nat. Rev. Immunol. 4 (6) (2004) 469 478. [75] D.F. Moyano, M. Goldsmith, D.J. Solfiell, D. Landesman-Milo, O.R. Miranda, D. Peer, et al., Nanoparticle hydrophobicity dictates immune response, J. Am. Chem. Soc. 134 (9) (2012) 3965 3967. [76] M.-A. Shahbazi, T.D. Fernández, E.M. Mäkilä, X. Le Guével, C. Mayorga, M.H. Kaasalainen, et al., Surface chemistry dependent immunostimulative potential of porous silicon nanoplatforms, Biomaterials 35 (33) (2014) 9224 9235. [77] E.M.V. Hoek, G.K. Agarwal, Extended DLVO interactions between spherical particles and rough surfaces, J. Colloid Interface Sci. 298 (1) (2006) 50 58. [78] L. Circelli, M. Tornesello, F.M. Buonaguro, L. Buonaguro, Use of adjuvants for immunotherapy, Hum. Vacin. Immunother. 13 (8) (2017) 1774 1777. [79] B. Temizoz, E. Kuroda, K.J. Ishii, Vaccine adjuvants as potential cancer immunotherapeutics, Int. Immunol. 28 (7) (2016) 329 338. [80] S. Sarkar, A.C.D. Salyer, K.A. Wall, S.J. Sucheck, Synthesis and immunological evaluation of a MUC1 glycopeptide incorporated into l-rhamnose displaying liposomes, Bioconjug. Chem. 24 (3) (2013) 363 375. [81] L. Amiset, L. Fend, T. Gatard-Scheikl, K. Rittner, V. Duong, R. Rooke, et al., TLR2 ligation protects effector T cells from regulatory T-cell mediated suppression and repolarizes T helper responses following MVA-based cancer immunotherapy, Oncoimmunology 1 (8) (2012) 1271 1280. [82] C.J. Hertz, S.M. Kiertscher, P.J. Godowski, D.A. Bouis, M.V. Norgard, M.D. Roth, et al., Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2, J. Immunol. (Baltimore, MD. 1950) 166 (4) (2001) 2444 2450. [83] S. Borsutzky, K. Kretschmer, P.D. Becker, P.F. Muhlradt, C.J. Kirschning, S. Weiss, et al., The mucosal adjuvant macrophage-activating lipopeptide-2 directly stimulates B lymphocytes via the TLR2 without the need of accessory cells, J. Immunol. (Baltimore, MD. : 1950) 174 (10) (2005) 6308 6313. [84] M.L. Salem, S.A. El-Naggar, A. Kadima, W.E. Gillanders, D.J. Cole, The adjuvant effects of the Toll-like receptor 3 ligand polyinosinic-cytidylic acid poly (I:C) on antigen-specific CD81 T cell responses are partially dependent on NK cells with the induction of a beneficial cytokine milieu, Vaccine 24 (24) (2006) 5119 5132. [85] C. Stahl-Hennig, M. Eisenblatter, E. Jasny, T. Rzehak, K. Tenner-Racz, C. Trumpfheller, et al., Synthetic double-stranded RNAs are adjuvants for the induction of T helper 1 and humoral immune responses to human papillomavirus in rhesus macaques, PLoS Pathog. 5 (4) (2009) e1000373.

Chapter 10 • Advanced Nanovaccines for Immunotherapy Applications

257

[86] O. Pfaar, D. Cazan, L. Klimek, D. Larenas-Linnemann, M.A. Calderon, Adjuvants for immunotherapy, Curr. Opin. Allergy Clin. Immunol. 12 (6) (2012) 648 657. [87] V. Mata-Haro, C. Cekic, M. Martin, P.M. Chilton, C.R. Casella, T.C. Mitchell, The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4, Science 316 (5831) (2007) 1628. [88] Z. Zhang, S. Tongchusak, Y. Mizukami, Y.J. Kang, T. Ioji, M. Touma, et al., Induction of anti-tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery, Biomaterials 32 (14) (2011) 3666 3678. [89] P. Elamanchili, C.M. Lutsiak, S. Hamdy, M. Diwan, J. Samuel, Pathogen-mimicking” nanoparticles for vaccine delivery to dendritic cells, J. Immunother. (Hagerstown, MD: 1997) 30 (4) (2007) 378 395. [90] R.E. Royal, L.M. Vence, T. Wray, J.N. Cormier, J.E. Lee, J.E. Gershenwald, et al., A Toll-like receptor agonist to drive melanoma regression as a vaccination adjuvant or by direct tumor application, J. Clin. Oncol. 35 (15_suppl) (2017) 9582. [91] S. Adams, D.W. O’Neill, D. Nonaka, E. Hardin, L. Chiriboga, K. Siu, et al., Immunization of malignant melanoma patients with full-length NY-ESO-1 protein using TLR7 agonist imiquimod as vaccine adjuvant, J. Immunol. (Baltimore, MD: 1950) 181 (1) (2008) 776 784. [92] C. Bode, G. Zhao, F. Steinhagen, T. Kinjo, D.M. Klinman, CpG DNA as a vaccine adjuvant, Exp. Rev. Vac. 10 (4) (2011) 499 511. [93] D.M. Klinman, Immunotherapeutic uses of CpG oligodeoxynucleotides, Nat. Rev. Immunol. 4 (4) (2004) 249 258. [94] S. Modak, G. Koehne, A. Vickers, R.J. O’Reilly, N.K. Cheung, Rituximab therapy of lymphoma is enhanced by orally administered (1-- . 3),(1-- . 4)-D-beta-glucan, Leuk. Res. 29 (6) (2005) 679 683. [95] Y. Masuda, H. Inoue, H. Ohta, A. Miyake, M. Konishi, H. Nanba, Oral administration of soluble betaglucans extracted from Grifola frondosa induces systemic antitumor immune response and decreases immunosuppression in tumor-bearing mice, Int. J. Cancer 133 (1) (2013) 108 119. [96] J. Fu, D.B. Kanne, M. Leong, L.H. Glickman, S.M. McWhirter, E. Lemmens, et al., STING agonist formulated cancer vaccines can cure established tumors resistant to PD-1 blockade, Sci. Transl. Med. 7 (283) (2015) 283ra52. [97] T.W. Dubensky, S.G. Reed, Adjuvants for cancer vaccines, Semin. Immunol. 22 (3) (2010) 155 161. [98] K. Takeda, S. Akira, TLR signaling pathways, Semin. Immunol. 16 (1) (2004) 3 9. [99] J.A. Hubbell, S.N. Thomas, M.A. Swartz, Materials engineering for immunomodulation, Nature 462 (2009) 449. [100] H. Yan, T. Kamiya, P. Suabjakyong, N.M. Tsuji, Targeting C-type lectin receptors for cancer immunity, Front. Immunol. 6 (2015) 408. [101] R.L. Sabado, N. Bhardwaj, Directing dendritic cell immunotherapy towards successful cancer treatment, Immunotherapy 2 (1) (2010) 37 56. [102] L.C. Bonifaz, D.P. Bonnyay, A. Charalambous, D.I. Darguste, S. Fujii, H. Soares, et al., In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination, J. Exp. Med. 199 (6) (2004) 815 824. [103] L. Corrales, T.F. Gajewski, Molecular pathways: targeting the stimulator of interferon genes (STING) in the immunotherapy of cancer, Clin. Cancer Res. 21 (21) (2015) 4774 4779. [104] L. Liu, P. Ma, H. Wang, C. Zhang, H. Sun, C. Wang, et al., Immune responses to vaccines delivered by encapsulation into and/or adsorption onto cationic lipid-PLGA hybrid nanoparticles, J. Controlled Release 225 (2016) 230 239. [105] W. Zhang, L. Wang, Y. Liu, X. Chen, Q. Liu, J. Jia, et al., Immune responses to vaccines involving a combined antigen nanoparticle mixture and nanoparticle-encapsulated antigen formulation, Biomaterials 35 (23) (2014) 6086 6097.

258

THERANOSTIC BIONANOMATERIALS

[106] D. Boraschi, P. Italiani, Model validity in nanoimmunosafety: advantages and disadvantages of in vivo vs in vitro models, and human vs animal models, Curr. Bionanotechnol. 2 (2) (2016) 71 76. [107] T. Laaksonen, H. Santos, H. Vihola, J. Salonen, J. Riikonen, T. Heikkilä, et al., Failure of MTT as a toxicity testing agent for mesoporous silicon microparticles, Chem. Res. Toxicol. 20 (12) (2007) 1913 1918. [108] M.A. Dobrovolskaia, S.E. McNeil, Immunological properties of engineered nanomaterials, Nat. Nanotechnol. 2 (8) (2007) 469 478. [109] M.A. Dobrovolskaia, D.R. Germolec, J.L. Weaver, Evaluation of nanoparticle immunotoxicity, Nat. Nanotechnol. 4 (7) (2009) 411 414. [110] M.A. Shahbazi, M. Hamidi, E.M. Mäkilä, H. Zhang, P.V. Almeida, M. Kaasalainen, et al., The mechanisms of surface chemistry effects of mesoporous silicon nanoparticles on immunotoxicity and biocompatibility, Biomaterials 34 (31) (2013) 7776 7789. [111] L. Shang, K. Nienhaus, G.U. Nienhaus, Engineered nanoparticles interacting with cells: size matters, J. Nanobiotechnol. 12 (2014) 5. [112] T. dos Santos, J. Varela, I. Lynch, A. Salvati, K.A. Dawson, Quantitative assessment of the comparative nanoparticle-uptake efficiency of a range of cell lines, Small 7 (23) (2011) 3341 3349. [113] G. Caracciolo, S. Palchetti, V. Colapicchioni, L. Digiacomo, D. Pozzi, A.L. Capriotti, et al., Stealth effect of biomolecular corona on nanoparticle uptake by immune cells, Langmuir 31 (39) (2015) 10764 10773. [114] V.L. MacKay, E.E. Moore, Immortalized Dendritic Cells, Google Patents, 1997. [115] N.K. Kunda, I.M. Alfagih, E.N. Miyaji, D.B. Figueiredo, V.M. Goncalves, D.M. Ferreira, et al., Pulmonary dry powder vaccine of pneumococcal antigen loaded nanoparticles, Int. J. Pharm. 495 (2) (2015) 903 912. [116] R. Kuai, L.J. Ochyl, K.S. Bahjat, A. Schwendeman, J.J. Moon, Designer vaccine nanodiscs for personalized cancer immunotherapy, Nat. Mater. 16 (4) (2017) 489 496. [117] L. Qiu, M. Valente, Y. Dolen, E. Jäger, M.T. Beest, L. Zheng, et al., Endolysosomal-escape nanovaccines through adjuvant-induced tumor antigen assembly for enhanced effector CD81 T cell activation, Small 14 (15) (2018) 1703539. [118] M.A. Shetab Boushehri, M.M.A. Abdel-Mottaleb, A. Beduneau, Y. Pellequer, A. Lamprecht, A nanoparticle-based approach to improve the outcome of cancer active immunotherapy with lipopolysaccharides, Drug Deliv. 25 (1) (2018) 1414 1425. [119] S.K. Datta, V. Redecke, K.R. Prilliman, K. Takabayashi, M. Corr, T. Tallant, et al., A subset of Toll-like receptor ligands induces cross-presentation by bone marrow-derived dendritic cells, J. Immunol. 170 (8) (2003) 4102 4110. [120] C. Zhang, G. Shi, J. Zhang, H. Song, J. Niu, S. Shi, et al., Targeted antigen delivery to dendritic cell via functionalized alginate nanoparticles for cancer immunotherapy, J. Controlled Release 256 (2017) 170 181. [121] M. Luo, H. Wang, Z. Wang, H. Cai, Z. Lu, Y. Li, et al., A STING-activating nanovaccine for cancer immunotherapy, Nat. Nanotechnol. 12 (7) (2017) 648 654. [122] A. Yeste, M.C. Takenaka, I.D. Mascanfroni, M. Nadeau, J.E. Kenison, B. Patel, et al., Tolerogenic nanoparticles inhibit T cell mediated autoimmunity through SOCS2, Sci. Signal 9 (433) (2016) ra61. [123] R.M. Pearson, L.M. Casey, K.R. Hughes, L.Z. Wang, M.G. North, D.R. Getts, et al., Controlled delivery of single or multiple antigens in tolerogenic nanoparticles using peptide-polymer bioconjugates, Mol. Ther. 25 (7) (2017) 1655 1664. [124] Y. Li, P. Italiani, E. Casals, D. Valkenborg, I. Mertens, G. Baggerman, et al., Assessing the immunosafety of engineered nanoparticles with a novel in vitro model based on human primary monocytes, ACS Appl. Mater. Interfaces 8 (42) (2016) 28437 28447.

Chapter 10 • Advanced Nanovaccines for Immunotherapy Applications

259

[125] F. Fontana, M.A. Shahbazi, D. Liu, H. Zhang, E. Mäkilä, J. Salonen, et al., Multistaged nanovaccines based on porous silicon@acetalated dextran@cancer cell membrane for cancer immunotherapy, Adv. Mater. 29 (7) (2017). [126] L.E. Higdon, K. Lee, Q. Tang, J.S. Maltzman, Virtual global transplant laboratory standard operating procedures for blood collection, PBMC isolation, and storage, Transpl. Direct 2 (9) (2016) e101. [127] H.W. Grievink, T. Luisman, C. Kluft, M. Moerland, K.E. Malone, Comparison of three isolation techniques for human peripheral blood mononuclear cells: cell recovery and viability, population composition, and cell functionality, Biopreserv. Biobanking 14 (5) (2016) 410 415. [128] A. Mohammadi, J. Mehrzad, M. Mahmoudi, M. Schneider, A. Haghparast, Effect of culture and maturation on human monocyte-derived dendritic cell surface markers, necrosis and antigen binding, Biotech. Histochem. 90 (6) (2015) 445 452. [129] T. Bauleth-Ramos, M.A. Shahbazi, D. Liu, F. Fontana, A. Correia, P. Figueiredo, et al., Nutlin-3a and cytokine co-loaded spermine-modified acetalated dextran nanoparticles for cancer chemo-immunotherapy, Adv. Funct. Mater. 27 (42) (2017) 1703303. [130] B.W. Neun, M.A. Dobrovolskaia, Analysis of nanoparticle-adjuvant properties in vivo, Methods Mol. Biol. 2018 (1682) 189 195. [131] B.M. Holzapfel, F. Wagner, L. Thibaudeau, J.P. Levesque, D.W. Hutmacher, Concise review: humanized models of tumor immunology in the 21st century: convergence of cancer research and tissue engineering, Stem Cells 33 (6) (2015) 1696 1704. [132] B.A. Ruggeri, F. Camp, S. Miknyoczki, Animal models of disease: pre-clinical animal models of cancer and their applications and utility in drug discovery, Biochem. Pharmacol. 87 (1) (2014) 150 161. [133] X. Guan, J. Chen, Y. Hu, L. Lin, P. Sun, H. Tian, et al., Highly enhanced cancer immunotherapy by combining nanovaccine with hyaluronidase, Biomaterials 171 (2018) 198 206. [134] A.V. Kroll, R.H. Fang, Y. Jiang, J. Zhou, X. Wei, C.L. Yu, et al., Nanoparticulate delivery of cancer cell membrane elicits multiantigenic antitumor immunity, Adv. Mater. 29 (47) (2017). [135] Y. Guo, D. Wang, Q. Song, T. Wu, X. Zhuang, Y. Bao, et al., Erythrocyte membrane-enveloped polymeric nanoparticles as nanovaccine for induction of antitumor immunity against melanoma, ACS Nano 9 (7) (2015) 6918 6933. [136] A.I. Bocanegra Gondan, A. Ruiz-de-Angulo, A. Zabaleta, N. Gomez Blanco, B.M. Cobaleda-Siles, M.J. Garcia-Granda, et al., Effective cancer immunotherapy in mice by polyIC-imiquimod complexes and engineered magnetic nanoparticles, Biomaterials 170 (2018) 95 115. [137] A. Kellar, C. Egan, D. Morris, Preclinical murine models for lung cancer: clinical trial applications, Biomed. Res. Int. 2015 (2015) 621324. [138] J. Zuo, M. Wen, M. Lei, X. Xiao, Z. Liu, PLGA-Der p1 vaccine inhibited tumor growth in a murine model of lung cancer, Arch. Med. Res. (2015). [139] G. Zhu, L. Mei, H.D. Vishwasrao, O. Jacobson, Z. Wang, Y. Liu, et al., Intertwining DNA-RNA nanocapsules loaded with tumor neoantigens as synergistic nanovaccines for cancer immunotherapy, Nat. Commun. 8 (1) (2017) 1482. [140] R. Yang, J. Xu, L. Xu, X. Sun, Q. Chen, Y. Zhao, et al., Cancer cell membrane-coated adjuvant nanoparticles with mannose modification for effective anticancer vaccination, ACS Nano 12 (6) (2018) 5121 5129. [141] P. Caplazi, M. Baca, K. Barck, R.A. Carano, J. DeVoss, W.P. Lee, et al., Mouse models of rheumatoid arthritis, Vet. Pathol. 52 (5) (2015) 819 826. [142] R. Heo, D.G. You, W. Um, K.Y. Choi, S. Jeon, J.-S. Park, et al., Dextran sulfate nanoparticles as a theranostic nanomedicine for rheumatoid arthritis, Biomaterials 131 (2017) 15 26. [143] E. Nogueira, A.C. Gomes, A. Preto, A. Cavaco-Paulo, Folate-targeted nanoparticles for rheumatoid arthritis therapy, Nanomedicine 12 (4) (2016) 1113 1126.

260

THERANOSTIC BIONANOMATERIALS

[144] H.J. Byeon, C. Lee, S. Lee, E.S. Lee, H.-G. Choi, E.-S. Park, et al., Pharmaceutical potential of tacrolimus-loaded albumin nanoparticles having targetability to rheumatoid arthritis tissues, Int. J. Pharm. 497 (1-2) (2016) 268 276. [145] M.M. Alam, H.S. Han, S. Sung, J.H. Kang, K.H. Sa, H. Al Faruque, et al., Endogenous inspired biomineral-installed hyaluronan nanoparticles as pH-responsive carrier of methotrexate for rheumatoid arthritis, J. Controlled Release 252 (2017) 62 72. [146] F. Fontana, S. Albertini, A. Correia, M. Kemell, R. Lindgren, E. Mäkilä, et al., Bioengineered porous silicon nanoparticles@ macrophages cell membrane as composite platforms for rheumatoid arthritis, Adv. Funct. Mater. 28 (22) (2018) 1801355. [147] C.S. Constantinescu, N. Farooqi, K. O’brien, B. Gran, Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS), Br. J. Pharmacol. 164 (4) (2011) 1079 1106. [148] K. Kirschbaum, J.K. Sonner, M.W. Zeller, K. Deumelandt, J. Bode, R. Sharma, et al., In vivo nanoparticle imaging of innate immune cells can serve as a marker of disease severity in a model of multiple sclerosis, Proc. Natl. Acad. Sci. U.S.A. 113 (46) (2016) 13227 13232. [149] E. Montes-Cobos, S. Ring, H.J. Fischer, J. Heck, J. Strauss, M. Schwaninger, et al., Targeted delivery of glucocorticoids to macrophages in a mouse model of multiple sclerosis using inorganic-organic hybrid nanoparticles, J. Controlled Release 245 (2017) 157 169. [150] M.L. Graham, H.J. Schuurman, Validity of animal models of type 1 diabetes, and strategies to enhance their utility in translational research, Eur. J. Pharmacol. 759 (2015) 221 230. [151] J.P. Martins, R. D’Auria, D. Liu, F. Fontana, M.P.A. Ferreira, A. Correia, et al., Engineered multifunctional albumin-decorated porous silicon nanoparticles for FcRn translocation of insulin, Small 14 (27) (2018) e1800462. [152] N. Shrestha, F. Araújo, M.A. Shahbazi, E. Mäkilä, M.J. Gomes, B. Herranz-Blanco, et al., Thiolation and cell-penetrating peptide surface functionalization of porous silicon nanoparticles for oral delivery of insulin, Adv. Funct. Mater. 26 (20) (2016) 3405 3416. [153] P. Serra, P. Santamaria, Nanoparticle-based approaches to immune tolerance for the treatment of autoimmune diseases, Eur. J. Immunol. 48 (5) (2018) 751 756. [154] X. Clemente-Casares, J. Blanco, P. Ambalavanan, J. Yamanouchi, S. Singha, C. Fandos, et al., Expanding antigen-specific regulatory networks to treat autoimmunity, Nature 530 (7591) (2016) 434 440. [155] R.A. Maldonado, R.A. LaMothe, J.D. Ferrari, A.H. Zhang, R.J. Rossi, P.N. Kolte, et al., Polymeric synthetic nanoparticles for the induction of antigen-specific immunological tolerance, Proc. Natl. Acad. Sci. U.S.A. 112 (2) (2015) E156 E165. [156] Z. Hunter, D.P. McCarthy, W.T. Yap, C.T. Harp, D.R. Getts, L.D. Shea, et al., A biodegradable nanoparticle platform for the induction of antigen-specific immune tolerance for treatment of autoimmune disease, ACS Nano 8 (3) (2014) 2148 2160.

11 Two-Dimensional Nanomaterials in Cancer Theranostics Jiuhai Wang, Mo Yang DEPART ME NT OF BIOMEDICAL ENGINEERING, THE HONG KONG POLYTECHNIC UN IVERSITY, HONG K ONG S.A.R., P.R. CHINA

11.1 Introduction The recent development of nanomaterials with highly controlled structures and interesting properties has created new opportunities for disease diagnosis and therapy, especially in oncology [1]. Materials at the nanoscale dimension are believed to have unique physicochemical properties which make them suitable for a variety of biomedical applications. Nanomaterials can be fabricated into various shapes and morphologies, such as spherical nanoparticles, nanorods, nanosheets, nanodots, nanotubes, nanowires, and nanocages, among others [2 4]. It is believed that the morphology of nanomaterials is highly associated with their biological performance, such as cellular uptake, cytotoxicity, biodistribution, and blood circulation time. Mesoporous nanostructures including silica nanoparticles and metalorganic frameworks (MOFs) have been extensively studied due to their extremely high surface area, which is favorable for loading guest molecules such as genes and drugs in cancer therapy and other biomedical applications [5,6]. Two-dimensional (2D) nanomaterials, an emerging class of nanomaterials that possess sheet-like structures with thickness of only a few atoms, are increasingly gaining interest for use in biomedical research [7,8]. Many biomedical applications of nanomaterials involve bioconjugation, where 2D nanomaterials are expected to be advantageous over conventional bulk materials due to their high surface-to-mass ratio. Graphene is a one-atom-thick film consisting of a single layer of carbon atoms arranged in a hexagonal lattice [9]. Its unprecedented properties including high electrical and thermal conductivity, ultrahigh roomtemperature carrier mobility, high optical transmittance, quantum Hall effect, and high Young’s modulus have brought about growing explorations of graphene and its derivates in the biomedical field [10]. For example, biocompatible graphene derivatives, such as graphene oxide (GO) and reduced GO (rGO), have been used for biosensing, chemodrug delivery, and photothermal therapy (PTT) [11 13]. Integration with other materials or chemical molecules enables graphene to be used as an excellent tool for tumor imaging and photodynamic therapy (PDT) [14]. In addition to graphene, a variety of other 2D materials,

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ranging from transition metal dichalcogenides (TMDs), MOFs, graphitic carbon nitride (g-C3N4), to black phosphorus (BP), layered double hydroxides (LDHs), and manganese dioxide (MnO2) nanosheets, have received considerable attention among biologists and material scientists in the last few years (Fig. 11 1). Their unique physical, chemical properties have facilitated their application in tumor therapy, such as gene and drug delivery, PTT, PDT, radiation therapy (RT), tissue engineering, biosensing, etc. [15 19]. In addition to cancer therapy, these 2D nanomaterials also have been demonstrated to be capable of enhancing the contrast for various imaging modalities including fluorescent imaging, magnetic resonance (MR) imaging (MRI), photoacoustic (PA) imaging, computed tomography (CT), positron emission tomography (PET), and radio-nuclide imaging [20 23]. In this chapter, a number of typical 2D nanomaterials, including graphene, TMDs, MOFs, g-C3N4, BP, and LDH will be introduced and the state-of-the-art progress of their theranostic applications will also be highlighted and summarized.

FIGURE 11–1 Summary of typical 2D nanomaterials. Reprinted with permission from H. Zhang, Ultrathin two-dimensional nanomaterials, ACS Nano 9 (10) (2015) 9451 9469 (https://pubs.acs.org/doi/abs/10.1021% 2Facsnano.5b05040. Any further permissions related to this material should be directed to the ACS). © 2015 American Chemical Society.

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11.2 Theranostic Payloads 11.2.1 Biomedical Imaging 11.2.1.1 Optical Imaging Optical imaging has been widely used in biomedical studies for a long time. Typically, visible light is used to excite dye molecules within a tissue, with a fluorescent emission at longer wavelength. Researchers developed a series of organic fluorescent dyes such as cyanine 5.5 (Cy5.5) and fluorescein isothiocyanate for both in vitro and in vivo optical imaging. These optical imaging agents, however, are usually excited with ultraviolet (UV) or visible light, which does not penetrate deep into tissue, resulting in limited use in the biomedical field. To solve this problem, two-photon near-infrared (NIR) molecules were developed for optical imaging because NIR light is minimally absorbed by hemoglobin and water so as to allow photons to penetrate for several centimeters within tissue [24,25]. In addition, NIR light is much safer and causes less damage to the human body compared with visible or UV light. To monitor a certain body site (usually a tumor site), the imaging contrast agent needs to be accumulated at the target site. A general, and simple, approach is to conjugate the fluorochrome to a ligand (can be nucleic acids, peptides, antibodies, etc.) that can specifically bind to the target. The fluorescent probes bind the targets retained at the target site and will not be cleared from the circulation. Chen et al. [26] developed NIR fluorescence-labeled folate probe for in vivo imaging of arthritis. Perez and coworkers [27] conjugated a functional apoptosis-initiating protein (cytochrome c) with a fluorescent tag indocyanine green and further with folic acid (FA) for targeted tumor imaging and cancer therapy. Fluorescent nanoparticles including quantum dots (QDs), fluorescence-labeled silica nanoparticles, and upconversion nanoparticles (UCNPs) constitute another family of imaging agent. Nie and coworkers developed multifunctional nanoparticle probes based on semiconductor QDs for cancer targeting and imaging in living animals [28]. The design involves encapsulating luminescent QDs with a polymer and conjugating this amphiphilic polymer with tumor-targeting molecules for the imaging and therapy of human prostate cancer. With different tagging of molecules, they achieved a sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions. UCNPs, a group of inorganic crystalline nanostructures, have received great scientific interest and been extensively used for biomedical imaging due to their unique NIR light absorption and visible luminescence properties. Unlike organic dyes which diffused rapidly in the circulation, UCNPs have excellent stability in a physiological environment, which makes them suitable for monitoring cell events in the human body. Schuck and coworkers reported small UCNPs with diameters under 10 nm for single-molecule imaging [21]. These sub-10-nm UCNPs required lower energy of excitation than those of conventional multiphoton molecules. Xing and coworkers developed a system based on photocaged UCNPs for in vitro and in vivo bioluminescence imaging [29].

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11.2.1.2 Magnetic Resonance Imaging MRI is a medical imaging technique that detects nuclear spin reorientations in a strong magnetic field to form pictures of the anatomy and the physiological processes of the body. Although X-ray can be well controlled in current medical settings, MRI is still considered a better choice than X-ray-based imaging techniques such as CT. MRI has been a powerful tool for monitoring the brain and central nervous system, such as imaging the event of amyloid-β peptide in the brains of Alzheimer’s patients, providing superior soft tissue contrast and spatial resolution. MRI contrast agents, which are used to improve the visibility of internal body structures, allow better interpretation in MRI. Superparamagnetic materials such as iron oxide nanoparticles (IONPs), gadolinium (Gd) chelates, and manganese dioxide (MnO2) are by far the most commonly used MRI contrast agents. Among them, Gd-DTPA, an FDA-approved agent, is the most widely used MR contrast agent in both the research lab and clinic. Gd chelates provide T1-weighted, or positive, signal enhancement. Superparamagnetic materials such as IONPs are clinically used for T2-weighted or negative signal enhancement [30]. Jia et al. used cyclic arginylglycylaspartic acid c(RGD) conjugated ultrasmall Fe3O4 nanoparticles (B5 nm) as T1-weighted ultrasensitive contrast agents for the early detection of malignant tumors in liver cancer [31]. The RGD-modified T1-Fe3O4 nanoprobes exhibited a high r1 of 7.74 mM21 s21 and an ultralow r2/r1 of 2.8 at a magnetic field of 3 T and achieved significant improvement in the sensitivity of T1-weighted MRI. Liu and coworkers [32] prepared a novel hybrid nanosystem consisting of UCNPs and IONPs by a layer-by-layer self-assembly approach. UCNPs were first prepared and conjugated with ultrasmall superparamagnetic IONPs. This multifunctional nanoparticle allows MRI-guided externally controlled stem cell translocation and therapy in vivo. Lin and coworkers have developed Mn-based nanoscale metal-organic frameworks (NMOFs) and Gd31-containing NMOFs for T1-weighted contrast enhancement [20,33]. It is also reported that Gd31-containing NMOFs could also be used as T2-weighted contrast, which is not achievable using smallmolecule Gd chelates [20] (Fig. 11 2).

11.2.1.3 X-Ray Computed Tomography Imaging Since its introduction in the 1970s, CT has been a noninvasive tool for the diagnosis of many primary and metastatic tumors. CT images are obtained by rotating a low-energy X-ray source around the object to produce cross-sectional (tomographic) images to construct a three-dimensional (3D) image. CT image contrast is dependent on differential tissue absorption of X-rays. CT is often used to provide images of tissue anatomy and is increasingly being used in conjunction with PET, which increases sensitivity and specificity of detection in many types of cancer [34]. High Z number elements such as iodine, barium, and bismuth are frequently used for CT contrast enhancement in clinical settings to obtain images of soft tissues [35]. Iodinated molecules currently are the most widely used CT contrast agents because of their excellent capability in absorbing X-rays. However, nonspecific distribution, rapid renal clearance, and vascular permeation have rather limited their microvascular and targeting performance. To overcome these limitations, Kim et al. developed a new CT

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FIGURE 11–2 (A) T1-weighted MR images of PEGylated T1-Fe3O4 and RGD-modified T1-Fe3O4 at different Fe concentrations in water or 20% FBS solutions. Plots of (B) 1/T1 and (C) 1/T2 against Fe concentration of PEGylated T1-Fe3O4 and RGD-modified T1-Fe3O4 in water or 20% FBS solutions at 3.0 T. r1 and r2 were calculated from the slopes of the corresponding linear fits of the experimental data. (D) Relaxation properties of different test samples measured in (B) and (C). Reprinted with permission from Z. Jia, L. Song, F. Zang, J. Song, W. Zhang, C. Yan, et al., Active-target T1-weighted MR imaging of tiny hepatic tumor via RGD modified ultra-small Fe3O4 nanoprobes, Theranostics 6 (11) (2016) 1780 1791.

contrast agent based on gold nanoparticles [36]. They prepared uniform gold nanoparticles with size around 30 nm by general reduction of HAuCl4, followed by a coating with polyethylene glycol (PEG) to impart antibiofouling properties. This PEG-coated gold nanoparticle showed a 5.7 times higher attenuation than that of the current iodine-based CT contrast agent. In the meantime, the blood circulation time of PEG-coated gold nanoparticles (.4 h) is much longer than that of iodine-based CT contrast agent Ultravist (,10 min). Rabin et al. prepared polyvinylpyrrolidone (PVP)-coated bismuth sulfide nanoparticles by a two-step protocol and used them as an effective CT imaging agent [23]. PVP is a biocompatible polymer and helps prevent the uncoated Bi2S3 nanocrystals from aggregating at physiological pH and ionic strength. Furthermore, PVP inhibits the nonspecific protein binding to the surface of Bi2S3 nanocrystals, which causes rapid clearance through the reticuloendothelial system. This polymer Bi2S3 nanoparticle solved the confronting viscosity problems and exhibited longer vascular half-life in microvasculature imaging compared with other CT contrast agents. Wen et al. used ultrasmall biocompatible WO32x nanodots for multimodality imaging and combined therapy [37]. Because of the high atomic number and the strong X-ray attenuation capability of tungsten, WO32x nanodots served as a superior contrast agent for CT imaging. In addition, WO32x nanodots were also able to be used for PA imaging and PTT and PDT for tumors due to the localized surface plasmon resonance (LSPR) tunable in the NIR region.

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11.2.1.4 Positron Emission Tomography Imaging PET is a highly sensitive noninvasive nuclear imaging technique that enables visualization of metabolic processes in the body. Unlike anatomy-based imaging techniques such as CT and MR, PET images high-energy gamma-rays that are emitted from inside the subject. Isotopes labeled with the biological object emit a positron from its nucleus that collides with a nearby electron, producing two gamma-rays which will be detected by PET camera. 15O, 13N, 11C, and 18F are the most frequently used positron-emitting isotopes. However, many of these positron-emitting isotopes suffer from rather short half-lives (,100 min), which means that their production and use often require expensive on-site cyclotron and radiochemistry facilities [34]. Lin et al. developed a yolk shell Fe3O4@Au hybrid nanocomposite that consists of a Fe3O4 core, 64Cu isotopes, drug molecules, as well as a layer of gold for MR, PA, and PET multimodal imaging. Due to the thermosensitive polymer on the surface, this hybrid nanocomposite could also be used for chemotherapy based on thermal release triggered by NIR irradiation. Zhou et al. used 18F-labeled Gd31/Yb31/Er31-doped NaYF4 nanophosphors (NPs) that integrated radioactivity, magnetic, and upconversion luminescent (UCL) properties into one single system for multimodality PET, MRI, and laser scanning UCL imaging both in vitro and in vivo [38]. Chen and coworkers used a 64Cu-labeled photosensitizer (PS)functionalized GO to obtain a combined fluorescence/PET imaging guided PDT [22].

11.2.1.5 Photoacoustic Imaging PA imaging is a relatively new biomedical imaging modality based on the PA effect of light absorbers. A short-pulsed laser is usually applied to produce ultrasound in targeted tissue, where the laser energy generates heat and expends, creating an acoustic signal that can be reconstructed to show the distribution of optical absorption inside the tissue. Compared to other conventional optical imaging modalities which may suffer from poor penetration and relatively low resolution due to light scattering, PA imaging, on the other hand, provides multiscale, high-resolution, noninvasive imaging of structures deep in tissue. Shang et al. described small-scale core shell gold nanorod@MOF (GNR@MIL-88) nanoparticles with average diameter of 90 nm [39]. GNR@MIL-88 (Fe) nanoparticles exhibited high monodispersity and homogeneity in an aqueous solution. This nanocomposite served not only as a PA and CT contrast agent due to the gold nanorod core, but also as a T2-weighted MR contrast agent because of the outer shell of iron-based MOF structure. Wang et al. designed a plasmonic Au-nanocomposite based on mesoporous silica-coated AuNR for the contrast enhancement for PA and CT imaging [40]. In addition, with the incorporation of Gd chelates and anticancer drugs to the structure, this smart AuNR has also been as a T1-weighted MR contrast agent and for chemotherapy of breast tumor.

11.2.1.6 Others Ultrasound imaging is a rather simple, sensitive, and inexpensive imaging technique that has been widely used in the clinic laboratory. A microbubble, a small gas-filled microsphere that has specific acoustic properties, has been shown to be an ideal contrast agent in ultrasound imaging. Zheng and coworkers developed a porphyrinic bacteriochlorophyll lipid

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microbubble with perfluoropropane gas encapsulated [41]. The perfluoropropane gas provided ultrasound imaging contrast. Upon exposure to ultrasound, the microbubble burst and transformed to porphyrin nanoparticles which could be used for fluorescent imaging and PA imaging contrast agent. A gas-generating calcium carbonate (CaCO3) mineralized nanoparticle was proposed to generate bubbles and trigger the release of anticancer drugs in an acidic environment for ultrasound imaging and tumor therapy [42].

11.2.2 Therapeutics 11.2.2.1 Chemotherapy Cancer cells are different from normal cells in that they divide relentlessly, forming solid tumors that show malignant behaviors such as invasion and metastasis. The uncontrolled cell growth is enabled by fast and abnormal production of DNA in cancer cells. Chemotherapy is a type of cancer treatment that uses chemotherapeutic drugs to interfere with the process of DNA replication and suppress abnormal cell division, leading to an inhibition of tumor growth and metastasis. Nucleic acid synthesis is a fundamental and essential process during cell proliferation, particularly at the early phase of cell growth cycle. Some anticancer drugs, therefore, have been developed to halt this unlimited cell proliferation by blocking the formation of pre-DNA molecules. Examples of therapeutic drugs of this category include 6-mercaptopurine, 5-fluorouracil (5-FU), methotrexate (MTX), and hydroxyurea (hydroxycarbamide). MTX participates in the inhibition of conversion of FA to tetrahydrofolic acids, leading to a low production of nucleic acids and proteins and blockage of cell division [43]. 5-FU is an inhibitor of thymidylate synthase that prevents nucleotide synthesis and arrests cell division. 5-FU can arrest unlimited proliferation of cancer cells and also lead to production of faulty rRNA [44]. Other drugs stop DNA replication by inhibiting the activity of telomerase or eliminating the telomeric DNA. Telomerase is a reverse transcriptase enzyme that presents in normal stem cells and most cancer cells to elongate telomeres and enables cells to divide. Drugs of this kind include doxorubicin (DOX), cisplatin, antibiotics, and etoposide. DOX is developed for the treatment of a variety of diseases including breast cancer, bladder cancer, Kaposi’s sarcoma, lymphoma, and acute lymphocytic leukemia by intercalating itself between nucleic acid bases, causing the cleavage of DNA. Etoposide has been approved for the treatment of lung cancer, choriocarcinoma, ovarian and testicular cancers, lymphoma, and acute myeloid leukemia. Etoposide does not intercalate into DNA but forms a ternary complex with DNA and topoisomerase II enzyme, causing DNA double-strand breaks, thereby inducing errors in DNA synthesis and apoptosis of cancer cells [45].

11.2.2.2 Photothermal Therapy PTT is a physicochemical therapy aiming at killing cancer cells by local hyperthermia achieved by employing a photo-absorbing agent, which converts optical energy into thermal energy upon irradiation with NIR light [46]. When an NIR light penetrates through tissues, photons are absorbed by tissue fluids and the energy is converted into heat [47]. It is known

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that thermoablation (46 C 56 C) causes tumor destruction by direct cell necrosis, whereas hyperthermia (41 C 45 C) induces intracellular protein damage, which is related to cellular survival and proliferation [48]. Numerous nanomaterials which exhibit strong optical absorption in the NIR regions have been developed for tumor-targeted PTT. These nanoscale particles that are intravenously administered to the body will preferentially accumulate in tumors areas due to the enhanced permeability and retention effect or functionalization of targeting molecules. With the introduction of NIR light, hyperthermia generated by the nanoparticles specifically heats the targeted tumor in an on-demand manner [49]. Plasmonic gold nanoparticles (AuNPs) exhibit unique photothermal properties due to LSPR. When shining with NIR light, AuNPs absorb photons and generate heat by photon electron and electron electron interactions for hyperthermia therapy [50]. Because of their strong absorption of NIR light, low toxicity, good biocompatibility, and chemical inertness, AuNPs have been considered as a promising nanoplatform for photothermal agents in cancer treatment [51]. Other gold-based nanostructures including gold nanorods (AuNRs) [52], hollow gold nanospheres (HAuNSs) [53], gold nanocages (AuNCs) [54], and carbon nanomaterials [55] also have been extensively studied for PTT in recently years (Fig. 11 3).

11.2.2.3 Photodynamic Therapy PDT is a clinically approved oncological intervention that involves administration of a PS followed by localized light irradiation of a specific wavelength. Three essential nontoxic components, PS, light, and oxygen, are necessary in PDT [56]. The PS converts the light energy to molecular oxygen to produce cytotoxic reactive oxygen species (ROS) that can rapidly oxidize key cellular macromolecules, leading to tumor cell ablation via apoptosis or necrosis [57]. Unlike chemotherapy that induces systemic toxicity to the whole body, and RT that cause damage to neighboring normal tissues, the individual components of PDT are nontoxic to biological systems, but together exhibit a cytotoxic effect through specific photochemical reactions [58]. PSs used for PDT should have certain properties such as high absorption between 600 nm and 800 nm, good water solubility, low toxicity in dark, and relatively

FIGURE 11–3 Schematic describing the principle of photothermal light to heat conversion by plasmonic nanostructures. Reprinted with permission from J.A. Webb, R. Bardhan, Emerging advances in nanomedicine with engineered gold nanostructures, Nanoscale 6 (5) (2014) 2502 2530.

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rapid clearance from normal tissues, among others. Light of short wavelength (,600 nm) cannot penetrate to deep tissue, whereas light at wavelength longer than 800 nm has insufficient energy to convert oxygen to its singlet state and to form a substantial yield of ROS. Porphyrins are FDA-approved and, so far, the most widely employed PSs are used for cancer PDT in the clinic. However, their photoabsorption at relatively short wavelength (B630 nm) confines their application to only a few cancer treatments. To improve this, a class of second-generation PSs that is based on porphyrin or chlorine structure has been discovered with absorption wavelengths between 635 and 762 nm [59].

11.2.2.4 Other Therapies Gene therapy is designed to transport a therapeutic genetic material (DNA or RNA) into specific cells of a patient to correct abnormal genes due to mutations [60]. If a mutant gene causes damage or loss of a necessary protein, the delivery of a normal copy of gene to the cell would be able to restore the function of that protein. Gene therapy was designed for specific treatments of numerous gene-related diseases such as cancer, cardiovascular disease, neurodegenerative disorders, and infectious disease. A number of different viruses have been developed as ideal vectors for the specific delivery of transgenes to the tissue or organ of interest because they can stably propagate in the cell culture [61]. In recent years, nanosized particles have attracted much attention in gene therapy. Poly(lactic-co-glycolic acid) (PLGA), among other biodegradable polymers, has been explored as a gene vector due to its high stability and ability to protect DNA from degradation during circulation in vivo [62]. Antibody-based therapy is another powerful and effective therapeutic option for hematological malignancies and solid tumors. Such a strategy involves delivering a specific antibodyconjugated drug that targets a specific antigen so as to alter its function [63]. Tumor antigens that have been successfully targeted include epidermal growth factor receptor, ERBB2, vascular endothelial growth factor, cytotoxic T lymphocyte-associated antigen 4 (CTLA4), CD20, CD30, and CD52.

11.3 Theranostic Two-Dimensional Nanomaterials 2D nanomaterials are attracting dramatically increased interest in the biomedical field, particularly tumor in theranostics. They are different from other types of nanomaterials, such as zero-dimensional (0D) nanoparticles, one-dimensional (1D) nanowires, and 3D materials or their bulk counterparts in terms of physical and chemical properties. There are several unique characteristics of 2D nanomaterials compared to their counterparts with other dimensionalities, including greatly compelling electronic properties, maximum mechanical flexibility and optical transparency, ultrahigh specific surface area, etc. These unique properties make 2D nanomaterials appealing candidates for the development of electronic biomedical devices or a highly favorable tool for drug delivery.

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11.3.1 Graphene and Its Derivatives Graphene is a term that describes a 2D monolayer of sp2 carbon atoms with a hexagonal packed structure [64]. Graphene and its derivatives, such as GO and rGO, have become the most exciting topics in the fields of material science, physics, and chemistry over the past decade and have been enormously investigated in recent years. This unique nanomaterial consists of only one plain layer of carbon atoms that are tightly arranged in a 2D honeycomb lattice, exhibiting unprecedented properties such as high electrical and thermal conductivity, ultrahigh room-temperature carrier mobility, high optical transmittance, quantum Hall effect, and high strength, which have fascinated the scientific community. Due to these unique physical, electronic, and chemical properties, graphene and its derivatives have been applied to a wide variety of biomedical applications including biomedical imaging, drug delivery, tissue engineering, and biosensors, among others.

11.3.1.1 Gene and Drug Delivery Although chemotherapy has been widely used in clinical settings, it appears that chemotherapeutic agents are not specific to cancer cells. This nonspecificity can cause severe damage to proliferating normal cells, including skin, hair, gastrointestinal, and bone marrow cells. Gene therapy, on the other hand, uses genes as medicine that involves the delivery of a therapeutic gene (DNA and RNA) into cells to repair gene defects inside the patient’s body. However, using gene therapy to cure disease is still challenging and faces difficulties in clinical translation. The major obstacle is that it is difficult to develop nonviral-based safe and efficient gene-delivery vehicles. Graphene is reported to have strong affinity with single-stranded DNA (ssDNA), while having a weak interaction with double-stranded DNA (dsDNA) molecules. Zhang et al. developed a novel gene-delivery system based on polyethylenimine branch modified graphene oxide (GO-PEI) [65]. GO-PEI exhibited excellent ability to carry plasmid DNA through an electrostatic interaction due to the cationic PEI on the surface. The fluorescence tracking revealed that the GO-PEI successfully delivered the gene into cells and cargo plasmid DNA was eventually localized in nucleus. Li and colleagues reported a chitosan (CS)-functionalized nanographene oxide (NGO-CS) nanocomplex constructed using an amide linkage, which displayed good solubility in acidic and neutral aqueous solutions [66]. In this work, an aromatic, water-insoluble anticancer drug, camptothecin (CPT), was attached to NGO-CS via van der Waals interactions. Meanwhile, NGO-CS also functioned as an ideal candidate for the delivery of plasmid DNA due to the excellent transfection efficiency and relatively low cytotoxicity of CS. Another novel graphene nanoplatform functionalized with polyamidoamine (PAMAM) dendrimer and oleic acid was reported as a biocompatible and efficient gene-delivery vector [67]. PAMAM was modified to the GO based on 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) chemistry. Positively charged primary amine groups in PAMAM dendrimers absorbed negatively charged DNA to form a GO-PAMAM-DNA complex and transported the DNA into a wide

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number of cell types. Compared with nondegradable PET, PAMAM exhibited excellent biocompatibility and minimal cytotoxicity. For drug delivery, a biocompatible and stimuli-responsive polymer, poly(N-isopropylacrylamide) (PNIPAM) functionalized GO (GO-PNIPAM) complex was introduced to deliver a water-insoluble drug, CPT, to cancer cells [68]. GO-PNIPAM exhibited a high CPT drug loading ratio of 18.5 wt.%. The in vitro test revealed that 16.9% and 19.4% of CPT were released from the nanocarrier in 72 h at 37 C in water and phosphate-buffered saline (PBS), respectively, demonstrating that GO-PNIPAM is a superior drug-delivery system for a variety of biomedical applications. A gelatin-functionalized graphene nanosheet with superior biocompatibility and physiological stability was developed and employed as an effective carrier to transport fluorescence probe rhodamine 6G (R6G) and anticancer drug DOX into MCF-7 cells to achieve simultaneous cell imaging and tumor chemotherapy [14].

11.3.1.2 Graphene as a Phototherapeutic Agent Because of its ultrathin 2D structure, graphene has an ultrahigh surface area available for efficient drug loading and functionalization on both sides. High NIR absorbance makes GO one of the best candidates for PTT. Zhang et al. developed a combinational therapeutic strategy for tumor based on a doxorubicin-loaded PEGylated nanographene oxide (NGO-PEG-DOX) [69]. In this nanoplatform, nanosized PEGylated GO (NGO-PEG) loaded with DOX and a layer of FA can specifically deliver both heat and anticancer drug to the tumor region to achieve simultaneous chemotherapy and PTT. Liu and coworkers designed a Chlorin e6 (Ce6)-loaded GO-PEG (GO-PEG-Ce6) via supramolecular π π stacking for photothermally enhanced PDT [11]. This nanocomplex provides dramatically improved photodynamic efficacy due to increased cellular uptake of Ce6 enhanced by a mild photothermal treatment. Tae et al. used a polymer-modified NGO complexed with methylene blue (MB), a positively charged hydrophilic PS, for combined PDT PTT of cancer. NGO was not only used as a photothermal agent, but also played a role as a vehicle for the delivery of MB to cells. The release of MB from NGO was triggered by acidic conditions at the tumor site [12]. Dai et al. developed a single-layered nano-rGO (NrGO) functionalized noncovalently with amphiphilic PEGylated polymer chains, resulting in a sixfold higher NIR absorption than nonreduced NGO [13]. With the linking of tumor-targeting peptide Arg-Gly-Asp (RGD), this NrGO-RGD could selectively target the U87MG cancer cells and exhibited effective photoablation of cells under NIR light. In addition to tumor therapy, graphene also has been used for the treatment of Alzheimer’s disease. Li et al. utilized thioflavin-S (ThS)-modified GO to locally dissociate the amyloid-β aggregates to decrease their neurotoxicity in the brain [70]. GO-ThS can selectively capture the amyloid-β aggregates and thermally break down the fibrillar forms under NIR light irradiation. This photothermal strategy is superior to conventional methods such as chemotherapy and radiotherapy because it functions locally and has minimal side effects. Furthermore, the nanosized nature enables GO-ThS to cross the blood brain barrier, and thus is suitable for the treatment of brain diseases.

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11.3.2 Transition Metal Dichalcogenides 2D TMD is a class of layered material consists of over 40 chemical compounds with a bandgap around 1 2 eV. TMDs have the general chemical formula MX2, where M is transition metal (e.g., Mo, W) and X is a chalcogen (e.g., S, Se, or Te) [71]. Atoms in a single layer are covalently bonded to each other and different layers can be held together via van der Waals forces. Layered TMDs show a wide range of electronic, optical, mechanical, chemical, and thermal properties that have been studied by researchers for decades [72]. In addition to their application in catalysis, energy storage, sensing, and electronic devices, 2D TMDs have been extensively studied for biological applications due to their versatility, controllability, and relatively high biocompatibility [73,74].

11.3.2.1 Transition Metal Dichalcogenides as Imaging Agents Among all TMD materials, molybdenum disulfide (MoS 2) has drawn much attention and been widely used in the biomedical field by both chemists and biologists due to its high physiological stability and superior biocompatibility. MoS2 has a strong PA effect as it can absorb optical energy of long wavelength (.700 nm) to create reflected ultrasound signals through thermoelastic expansion. An IONP-decorated 2D MoS2 nanosheet has been developed for multimodal imaging of cancer [75]. IONP was self-assembled on the surface of MoS2 nanosheets via a sulfur chemistry occurring on the defect sites of MoS2. 64 Cu, a commonly used PET radioisotope, was further adsorbed onto the surface of MoS2. IONP and MoS2 allowed this nanoplatform to be used for MRI and PA imaging, while 64Cu enabled PET imaging in the meantime. Chen and coworkers prepared a 2D MoS2/Bi2S3-PEG nanocomposite via a one-pot synthesis for CT and PA imaging due to the high X-ray attenuation of bismuth and PA effect of MoS2 [76]. Chen et al. demonstrated a single-layered MoS2 nanosheet with excellent biocompatibility and amplified PA effect for highly sensitive PA imaging of orthotopic brain tumors [77]. This study showed that the single-layered MoS2 has strong PA signal under NIR light and highly efficient tumor retention in vivo. Similar to MoS2, WS2 also has been used as an effective imaging probe for many imaging modalities. Due to the strong X-ray attenuation ability and high NIR optical absorbance of WS2, Liu and colleagues successfully utilized WS2PEG nanosheets for CT and PA imaging [18]. More recently, WS2@PEI nanocomposites [78] and 89Zr-labeled WS2 nanosheets [79] were also developed for multimodal imagingguided tumor therapy. A bottom-up method for one-pot synthesis of PVP-modified WS2 nanosheets (WS2-PVP) was designed based on the coordination of the PVP carbonyl group and the unoccupied orbital of tungsten [80]. The surface modification of PVP greatly improved the colloidal stability and biocompatibility of WS2 nanosheets. The strong X-ray attenuation and NIR absorbance of WS2-PVP enabled sensitive CT and PA imaging both in vitro and in vivo. Cheng et al. synthesized a Gd31-doped WS2 nanosheet (Gd-WS2) for CT, PA, and MR trimodal imaging as well as PTT [81].

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11.3.2.2 Transition Metal Dichalcogenides for Gene and Drug Delivery 2D nanomaterials like graphene have been extensively studied in recent years in the biomedical field. Due to the extremely large surface area of 2D nanomaterials that enables high cargo-loading capacity, they have been considered as a promising platform for gene and drug delivery. MoS2, one of the TMD family members, has drawn tremendous research interest in gene- and drug-delivery applications. Compared with other 2D materials like GO, MoS2 is easy to be functionalized as the exposed sulfur atoms on the surface provide plenty of bonding sites for sulfur-containing drug molecules to form disulfide bonds. These features lead MoS2 to be an ideal vehicle for effective gene and drug delivery. A novel concept of photothermally enhanced gene delivery recently has been proposed by several research groups. A single-layered MoS2 modified with two-polymer, PEG and PEI (MoS2-PEG-PEI), nanocomposite was developed by Kim et al. via disulfide bonding [82]. Cargo DNA was loaded to the MoS2-PEG-PEI through electrostatic interaction and shipped to targeted cells. Polymers were detached from the nanocomposite by photothermal generated by MoS2 with NIR irradiation, therefore triggering the release of DNA from MoS2-PEG-PEI-DNA complex. Kim’s group also developed a porous silica- and PEG-coated single-layered MoS2 nanocomposite loaded with DOX for NIR-responsive drug delivery [83]. Intracellular thermal release of DOX from an MoS2-based vehicle led to it being 30-fold more effective against hepatocarcinoma than free drugs in cancer treatment. Similar achievements have been made by Zhao and colleagues in CT and PET imaging-guided drug delivery and NIR photothermal release using CS-decorated single-layer MoS2 nanosheets [84].

11.3.2.3 Transition Metal Dichalcogenides as Photothermal Agents TMD nanomaterials, including MoS2, WS2, and MoSe2, have received much attention in the field of biomedicine and great progress has been made over the past few years. The electronic band structure of TMDs enables a strong optical absorption in the NIR region (B800 nm) which is minimally absorbed by biological tissue and excellent extinction coefficiency, allowing TMDs to be used as efficient phototherapeutic agents for the photothermal ablation of diseases such as cancer [85]. It is evidenced that the photothermal conversion efficiency of TMD, such as hydrophilic MoS 2 nanosheets, was comparable to the hydrophobic rGO, and the temperature of the aqueous solution of MoS2 at low concentration (B38 ppm) could be rapidly increased to above 40 C by a few minutes of low-power NIR irradiation (0.8 W/cm2). A cellular experiment showed significant HeLa cell deaths after coincubation with MoS2 and NIR irradiation (λ 5 800 nm, 20 min) [86]. Ultrathin MoS2 nanosheet was synthesized with controllable sizes by an oleum treatment exfoliation process by Zhao and colleagues [84]. The modification of cationic polysaccharide CS to the surface of MoS2 significantly improved its physiological stability and biocompatibility. The in vivo experiment showed that the temperature of the tumors that have taken in the MoS2-CS nanosheets increased quickly under 808 nm NIR irradiation (0.9 W/cm2) and reached a level (ΔT 5 22.5 C) which could effectively inhibit tumor growth.

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WS2 is another representative member of the TMD family. 2D WS2 has similar structure and physicochemical properties to MoS2 and exhibits promising photothermal conversion capability for PTT. An ultrathin WS2 nanosheet with an average thickness around 1.6 nm and an extinction coefficient about 23.8 Lg21 cm21 was synthesized by Liu and coworkers [18]. It was found that the WS2-PEG nanosheet could lead to an elevated temperature of the tumor to 65 C within 5-min irradiation by an 808 nm laser at a power density of 0.8 W/cm2. Imaging-guided PTT has also been achieved based on WS2 nanosheets. Zhao and colleagues used a WS2@poly(ethylene imine) nanoplatform for CT- and PA-guided PTT of cervical cancer [78]. The same group also reported a PS MB-incorporated WS2-BSA nanocomposite (WS2-BSA-MB) to be used as an effective therapeutic tool for simultaneous PDT and PTT of cancer [87] (Fig. 11 4).

11.3.3 Metal-Organic Frameworks MOFs, also called coordination polymers, are a class of inorganic and organic hybrid materials formed by self-assembly of metal ions or clusters and organic bridging ligands. Since the term “MOF” was first used by Yaghi in 1995, researchers have achieved significant progress in this field, from rational design and synthesis with predictable structures and functionality to applications such as gas storage/separation, catalysis, optical imaging, drug delivery, and sensing [88 90]. Various kinds of crystal structures in different space groups can be obtained by choosing different organic ligands and metals for the coordination. Even with the same metal and ligand, the structure and dimensionality of MOFs can vary with different synthesis method and auxiliary molecules. For example, Zhang and coworkers synthesized a series of ultrathin 2D MOF nanosheets using ligand tetrakis(4-carboxyphenyl)porphyrin (TCPP) and different metals (Zn, Cu, Cd, or Co) with the assistance of surfactant [91]. 2D MOFs with diverse physical/chemical properties, good biocompatibility, and suitable size have been considered as promising agents for disease theranostics. Liu and colleagues prepared a small and layered porphyrin-based 2D Zn-TCPP MOF nanodisk via a bottom-up method [92]. Due to the presence of fluorescent ligand TCPP in the structure, this Zn-TCPP MOF nanodisk emitted strong red fluorescence at 652 nm with an excitation wavelength at 415 nm. This unique optical property together with small size and excellent biocompatibility enables Zn-TCPP MOF nanodisks as a suitable candidate for fluorescence labeling cancer cells. A powerful Fe-MIL-53-NH2-FA-5-FAM MOF nanocomposite with drug encapsulated has been employed for simultaneous fluorescence/MRI as well as targeted antitumor drug delivery [93]. Due to the large surface area and multifunctional surface modification, this MOF nanocomposite exhibited good biocompatibility, tumorenhanced cellular uptake, strong cancer cell growth inhibition, excellent fluorescence imaging, and outstanding MRI capability. Gadolinium as a clinically accepted MRI agent also has been integrated into MOF for multimodal imaging for multiple studies. In particular, Gd (BDC)1.5(H2O)2 constructed by Gd metal clusters and BDC ligands were created as an effective MRI agent with r1 (longitudinal relaxivity) and r2 (transverse relaxivity) values ranging from 20.1 and 45.7 mM21 s21. It is hypothesized that the Gd31 release from the MOF is

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FIGURE 11–4 (A) A scheme showing MoS2-PEG/Ce6 nanosheets for Ce6 loading and combined photothermal and photodynamic therapy. (B) Photographs of MoS2 and MoS2-PEG in water or PBS. (C) UV vis NIR absorbance spectra of MoS2 and MoS2-PEG. IR thermal images (D) and photothermal heating curves (E) of MoS2-PEG solutions irradiated by an 808 nm laser (0.7 W/cm2) for 5 min. PBS, Phosphate-buffered saline. Reprinted with permission from T. Liu, C. Wang, W. Cui, H. Gong, C. Liang, X.Z. Shi, et al., Combined photothermal and photodynamic therapy delivered by PEGylated MoS2 nanosheets, Nanoscale 6 (19) (2014) 11219 11225. © Royal Society of Chemistry.

harmful to the body. Therefore a number of studies applied several other metal ions in the MOF synthesis including manganese and iron [94,95]. These types of MOFs showed a low cytotoxicity and superior biocompatibility, indicating a promising future for the development of MOF-based MR contrast agents. Recently, an ultrathin 2D zirconium MOF (PCN-222)

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nanosheet has been prepared by a solvothermal reaction of ZrCl4, TCPP, and formic acid (FA) in a solution of water and N,N-dimethylformamide [96]. It is reported that the layered structure of PCN-222 was formed through a pseudoassembly disassembly forming process with assistance of monocarboxylic acids (acetic acid, lauric acid, and oleic acid). Because of the 1O2 generation ability of porphyrin compounds in the MOF structure, this zirconiumporphyrinic PCN-222 has been studied and used for PDT of cancer.

11.3.4 Graphitic Carbon Nitride Graphitic carbon nitride (g-C3N4) is a relatively new type of carbon-based material that has been synthesized only in the last few years. Since first introduced in 2012, ultrathin g-C3N4 nanosheet has won tremendous attention among researchers because of its high intrinsic photoabsorption and photoresponsiveness, semiconductive properties, high stability under physiological conditions, and good biocompatibility. A g-C3N4 nanosheet with high photoluminescence (PL) intensity provides possibilities for its application in bioimaging. Xie and colleagues obtained ultrathin g-C3N4 nanosheet from bulk material through liquid-exfoliation process with a thickness around 2.5 nm [97]. This g-C3N4 nanosheet exhibited high PL intensity under UV light illumination. The confocal fluorescence microscopy indicated that g-C3N4 nanosheet was an excellent biocompatible agent for fluorescent imaging of cancer cells. Single-layered graphitic C3N4 QDs (g-C3N4 QDs) were also produced by the same group [98]. Porous g-C3N4 was first obtained by hydrothermal treatment with the aid of NH3
H2O, followed by a ultrasonication-assisted conversion from g-C3N4 nanosheet to single-layered g-C3N4 QDs. The single-layered g-C3N4 QDs are capable of being used as promising, safe, and economic fluorescent probes for in vivo and in vitro two-photon fluorescence imaging. Besides bioimaging, g-C3N4 nanosheet has also been shown to be able to carry drugs to the tumor site for cancer chemotherapy [98]. Owing to their high surface-to-volume ratio, g-C3N4 nanosheets have been reported to serve as a pH-responsive nanocarrier for DOX delivery with an ultrahigh loading capacity of 18,200 mg/g. DOX release from the g-C3N4 nanocarrier was successfully achieved via pH-responsive mechanism due to the low pH environment of the tumor area. The high photoabsorption nature and unique photocatalytic property also enable g-C3N4 nanosheets to be an effective PS for PDT. However, compared with the reported PSs that respond to NIR light, a g-C3N4 nanosheet with a relatively narrow band gap (2.7 eV) can only absorb UV light to produce 1O2 for PDT, which limits its application due to the strong tissue interference, low penetration depth, and possible skin damage from UV light. To tackle this problem, Lin and coworkers designed a hybrid UCNP@ g-C3N4 nanostructure to improve the ROS generation under excitation in a longer wavelength region [16]. UCNPs absorb photons in the NIR region and convert it to high-energy emission in short wavelength (UV and visible region). This UCNP@ g-C3N4 nanocomposite took the advantage of NIR light to excite g-C3N4 for ROS generation and achieved improved PDT efficacy for tumor therapy. Other platforms such as g-C3N4 embedded zeolitic-imadazolate framework-8 (g-C3N4@ZIF-8) [99], and Fe3O4-g-C3N4@mSiO2-PEG-RGD [100] also have been developed for photo chemo combinatory therapy.

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11.3.5 Black Phosphorus BP is a new member of the 2D material family. Since its first discovery, this metal-free layered semiconductor has received much interest due to the tunable layer-dependent bandgap of 0.3 2.0 eV and highly accurate optical-response properties. The distinct structure of BP nanosheets was constructed by puckered planes of phosphorus atoms via weak van der Waals forces. Similar to other layered materials such as graphite and TMDs, thin BP nanosheets with a few layers or even a monolayer can be obtained through mechanical and liquid exfoliation from bulk size [101]. BP has been found to exhibit unique properties, such as electronic conductivity, optical properties, thermoelectric properties, topological features, as well as its unusual mechanical behavior, leading to its extensive application in physical, chemical, and biomedical fields. More recently, studies have shown that BP can efficiently convert NIR light into heat with an excellent photostability, which makes BP a suitable nanotheranostic agent for PA imaging and PTT of cancer. Sun et al. developed a PEGylated BP obtained by means of high-energy mechanical milling technique and used it for PA imaging-guided PTT of breast cancer [102]. In vitro and in vivo studies showed that this nanotheranostic agent had excellent biocompatibility, photostability, and negligible toxicity. To improve the photothermal efficacy and biocompatibility of BP, Shao et al. have developed biodegradable PLGA-loaded BP (BPQDs/ PLGA) nanospheres by an emulsion method [15]. On the one hand, the hydrophobic PLGA layer served as a physical barrier to isolate BPQDs from oxygen and water to enhance the photothermal stability. On the other hand, PLGA helped control the degradation of BPQDs. The biocompatible and biodegradable BPQDs/PLGA nanospheres (NSs) showed excellent performance in PTT of cancer. Benefiting from the broad absorption across the UV and entire visible light region, BP nanosheets have been shown to be able to generate 1O2, suggesting its possible applications in catalysis and PDT. Studies in this area have shown that the ultrathin BP nanosheets exhibit significantly enhanced ability for 1O2 generation compared with their bulk counterpart, suggesting an efficient PDT therapeutic effect in the treatment of cancers [103]. A theranostic nanoprobe based on BP nanosheets was successfully designed with integration of Fe3O4 nanoparticles and plasmonic gold nanoparticles [104]. Owing to the photodynamic effect of BP, photothermal effect of Au, and MRI ability of Fe3O4 nanoparticles, this BPs@Au@Fe3O4 nanoplatform has been shown to have an MRI-guided photothermal and photodynamic function for tumor therapy with an accurate selective localization. Numerous research efforts have been devoted to the application of BP nanosheets in drug delivery. For example, small BP nanosheets (,20 nm) obtained by an ultrasonicationassisted exfoliation method were utilized for drug delivery as well as multiple color (blue and green) fluorescent imaging [105]. These small BP nanosheets were demonstrated to be stable in aqueous solution and not degraded after 10 days in PBS buffer solution. Chen et al. developed a BP-based drug-delivery system for synergistic PDT/PTT/chemotherapy of cancer [106]. DOX loading efficiency of BP nanosheet was measured to be 950% in weight, which is higher than that of other reported 2D material systems. Another novel nanoplatform that

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FIGURE 11–5 Singlet oxygen characterization. (A) Time-dependent absorption spectra of the 1,3Diphenylisobenzofuran (DPBF) with BP nanosheets in air. (B) Normalized absorbance of the DPBF in the presence of BP nanosheets in different conditions. (C) Electron Spin Resonance (ESR) spectra of BP nanosheets in the presence of TEMP in different conditions. Both the decomposing of DPBF and ESR spectra were carried out under Xe lamp with 600 nm cutoff filter. (D) 1O2 emission at B1270 nm induced by the commercial Rose Bengal and BP nanosheets in ethanol under excitation with a 530 nm light. Reprinted with permission from H. Wang, X.Z. Yang, W. Shao, S.C. Chen, J.F. Xie, X.D. Zhang, et al., Ultrathin black phosphorus nanosheets for efficient singlet oxygen generation, J. Am. Chem. Soc. 137 (35) (2015) 11376 11382. © 2015 American Chemical Society.

combines BP and hydrogel has been reported. Therapeutic drugs that encapsulated in the hydrogel can be released upon the hydrolysis of hydrogel triggered by the photothermal of BP nanosheets [107] (Fig. 11 5).

11.3.6 Other Two-Dimensional Nanomaterials LDHs, owing to their low toxicity, good cellular interaction, high drug loading capacity, and delivery efficacy, have been considered as novel and promising nanovehicles for drug delivery to cells. Most of the LDHs are formed by the positively charged brucite-like layers that are balanced by the interlayer anions. Therefore anionic pharmaceutical drugs such as 5-FU, MTX, ibuprofen, and nucleic acids are easy to be functionalized to the surface

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of LDHs [108 110]. Silica-coated Y2O3:Er31, Yb31 nanoparticles deposited with LDH layer intercalated with the drug 5-FU were designed by Chen et al. for tumor optical imaging guided chemotherapy [17]. The red upconversion fluorescence under the excitation of 980 nm laser allowed tracking of nanocarrier after being delivered to the cells. Similar studies have shown a high loading efficiency of DOX and codelivery of siRNA together with anticancer drugs for enhanced cancer therapy [111]. MnO2 nanosheet is another 2D material that has been extensively used in biomedical studies. PEGylation of MnO2 nanosheets improved their stability in physiological conditions, thus solving the issue of low-MRI performance of Mn-based contrast agents [112]. MnO2 nanosheets have been proved to be biocompatible and exhibit a unique break-up nature under mild acidic conditions. DOX could be released from MnO2 nanosheets after being delivered to the tumor site which has a lower pH than normal tissue. Another theranostic platform based on FA MnO2 ZnPc complex was developed by Kim et al. for tumor-targeted PDT and bioimaging [19].

11.4 Summary and Future Perspectives 2D material, without a doubt, has won great research interests and achieved significant progress in both fundamental studies and further biological applications over the past decade. Particularly, theranostic 2D nanomaterials with three functions, that is, imaging agent, therapeutic agent, and targeting moiety, have been under extensive investigation aimed at achieving early diagnosis as well as effective therapy of tumors with targeting ability. Medical imaging is considered as the most effective way to monitor tumor growth in clinical settings. Among all the imaging modalities, optical imaging, MRI, PA, CT, and PET are the commonly used techniques for tumor diagnosis. The development of 2D nanomaterials provides opportunities for the combination of multiple imaging modalities in a single nanoplatform to achieve multimodal imaging. Such nanoplatforms include iron-, gadolinium-, and manganese-based nanomaterials in combination with TMDs or AuNRs such as MIL-100, Mn(BDC)(H2O)2, Gd(BDC)1.5(H2O)2, ION-MoS2/WS2, MOF-AuNR, MnO2-AuNR, etc. With the functionalization of targeting molecules, these nanoprobes present a unique capability to be selectively localized at tumor site. 2D imaging nanoprobes with drug carrier ability, such as MnO2 nanosheets loaded with DOX in a high loading capacity, are suitable for imaging-guided chemotherapy of cancer. Some nanomaterials have an intrinsic therapeutic effect under certain conditions such as NIR light irradiation. Nanomaterials have long been utilized for disease therapeutics both in the laboratory and clinic. 2D nanomaterials, due to their extremely large surface area, are superior carriers for drug delivery compared with materials in other dimensions. There have been various 2D nanocarriers that have been developed to deliver anticancer drugs, such as MTX, DOX, and 5-FU, to cancer cells for tumor chemotherapy. A number of novel 2D nanomaterials with intrinsic photothermal and photodynamic properties are suitable for localized phototherapy of cancer. The combination of chemotherapeutic drugs and PTT/PDT agents lead to a photothermal release of drugs and unprecedented synergistic therapeutic efficacy. Recent attempts

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to apply the 2D nanomaterial in the synergistic therapy of Alzheimer’s disease seem to have a promising future. Despite the fact that great progress has been made in the development of theranostic 2D nanomaterials in the past decade, these agents are far from being applied clinically. Future efforts need to be made on the clinical translation of nanotheranostic agents, which means that these theranostic nanomaterials should be tested in animal models in order to fully understand the potential toxicity. Furthermore, theranostic nanocarriers for chemotherapeutic drug delivery need to have better targeting efficiency so as to avoid any harmfulness to the normal tissues. In order to meet the need of potent cancer therapies, newly designed 2D nanomaterials are expected to concentrate on addressing each specific case of cancer, achieving a personalized cancer treatment. By understanding the fundamentals of cancer development and the mechanism of the interactions between nanomaterials and cancer cells, chemists and material scientists would be able to accelerate the progress of applying 2D nanomaterials in the clinic.

References [1] C.E. Ashley, E.C. Carnes, G.K. Phillips, D. Padilla, P.N. Durfee, P.A. Brown, et al., The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers, Nat. Mater. 10 (5) (2011) 389 397. [2] Y.H. Bai, Y. Zhang, J.P. Zhang, Q.X. Mu, W.D. Zhang, E.R. Butch, et al., Repeated administrations of carbon nanotubes in male mice cause reversible testis damage without affecting fertility, Nat. Nanotechnol. 5 (9) (2010) 683 689. [3] Y. Piao, J. Kim, H.B. Na, D. Kim, J.S. Baek, M.K. Ko, et al., Wrap-bake-peel process for nanostructural transformation from beta-FeOOH nanorods to biocompatible iron oxide nanocapsules, Nat. Mater. 7 (3) (2008) 242 247. [4] M.S. Yavuz, Y.Y. Cheng, J.Y. Chen, C.M. Cobley, Q. Zhang, M. Rycenga, et al., Gold nanocages covered by smart polymers for controlled release with near-infrared light, Nat. Mater. 8 (12) (2009) 935 939. [5] F.Q. Tang, L.L. Li, D. Chen, Mesoporous silica nanoparticles: synthesis, biocompatibility and drug delivery, Adv. Mater. 24 (12) (2012) 1504 1534. [6] P. Horcajada, R. Gref, T. Baati, P.K. Allan, G. Maurin, P. Couvreur, et al., Metal-organic frameworks in biomedicine, Chem. Rev. 112 (2) (2012) 1232 1268. [7] C. Tan, X. Cao, X.J. Wu, Q. He, J. Yang, X. Zhang, et al., Recent advances in ultrathin two-dimensional nanomaterials, Chem. Rev. 117 (9) (2017) 6225 6331. [8] H. Zhang, Ultrathin two-dimensional nanomaterials, ACS Nano 9 (10) (2015) 9451 9469. [9] R.R. Nair, P. Blake, A.N. Grigorenko, K.S. Novoselov, T.J. Booth, T. Stauber, et al., Fine structure constant defines visual transparency of graphene, Science 320 (5881) (2008) 1308. [10] K. Yang, L.Z. Feng, X.Z. Shi, Z. Liu, Nano-graphene in biomedicine: theranostic applications, Chem. Soc. Rev. 42 (2) (2013) 530 547. [11] B. Tian, C. Wang, S. Zhang, L.Z. Feng, Z. Liu, Photothermally enhanced photodynamic therapy delivered by nano-graphene oxide, ACS Nano 5 (9) (2011) 7000 7009. [12] A. Sahu, W.I. Choi, J.H. Lee, G. Tae, Graphene oxide mediated delivery of methylene blue for combined photodynamic and photothermal therapy, Biomaterials 34 (26) (2013) 6239 6248.

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[13] J.T. Robinson, S.M. Tabakman, Y.Y. Liang, H.L. Wang, H.S. Casalongue, D. Vinh, et al., Ultrasmall reduced graphene oxide with high near-infrared absorbance for photothermal therapy, J. Am. Chem. Soc. 133 (17) (2011) 6825 6831. [14] K.P. Liu, J.J. Zhang, F.F. Cheng, T.T. Zheng, C.M. Wang, J.J. Zhu, Green and facile synthesis of highly biocompatible graphene nanosheets and its application for cellular imaging and drug delivery, J. Mater. Chem. 21 (32) (2011) 12034 12040. [15] J.D. Shao, H.H. Xie, H. Huang, Z.B. Li, Z.B. Sun, Y.H. Xu, et al., Biodegradable black phosphorus-based nanospheres for in vivo photothermal cancer therapy, Nat. Commun. 7 (2016) 12967. [16] L.L. Feng, F. He, B. Liu, G.X. Yang, S.L. Gai, P.P. Yang, et al., g-C3N4 coated upconversion nanoparticles for 808 nm near-infrared light triggered phototherapy and multiple imaging, Chem. Mater. 28 (21) (2016) 7935 7946. [17] C.P. Chen, L.K. Yee, H. Gong, Y. Zhang, R. Xu, A facile synthesis of strong near infrared fluorescent layered double hydroxide nanovehicles with an anticancer drug for tumor optical imaging and therapy, Nanoscale 5 (10) (2013) 4314 4320. [18] L. Cheng, J.J. Liu, X. Gu, H. Gong, X.Z. Shi, T. Liu, et al., PEGylated WS2 nanosheets as a multifunctional theranostic agent for in vivo dual-modal CT/photoacoustic imaging guided photothermal therapy, Adv. Mater. 26 (12) (2014) 1886 1893. [19] S. Kim, S.M. Ahn, J.S. Lee, T.S. Kim, D.H. Min, Functional manganese dioxide nanosheet for targeted photodynamic therapy and bioimaging in vitro and in vivo, 2D Mater. 4 (2) (2017) 025069. [20] W.J. Rieter, K.M.L. Taylor, H.Y. An, W.L. Lin, W.B. Lin, Nanoscale metal-organic frameworks as potential multimodal contrast enhancing agents, J. Am. Chem. Soc. 128 (28) (2006) 9024 9025. [21] D.J. Gargas, E.M. Chan, A.D. Ostrowski, S. Aloni, M.V.P. Altoe, E.S. Barnard, et al., Engineering bright sub-10-nm upconverting nanocrystals for single-molecule imaging, Nat. Nanotechnol. 9 (4) (2014) 300 305. [22] P.F. Rong, K. Yang, A. Srivastan, D.O. Kiesewetter, X.Y. Yue, F. Wang, et al., Photosensitizer loaded nano-graphene for multimodality imaging guided tumor photodynamic therapy, Theranostics 4 (3) (2014) 229 239. [23] O. Rabin, J.M. Perez, J. Grimm, G. Wojtkiewicz, R. Weissleder, An X-ray computed tomography imaging agent based on long-circulating bismuth sulphide nanoparticles, Nat. Mater. 5 (2) (2006) 118 122. [24] W.H. Lee, H. Lee, J.A. Kim, J.H. Choi, M.H. Cho, S.J. Jeon, et al., Two-photon absorption and nonlinear optical properties of octupolar molecules, J. Am. Chem. Soc. 123 (43) (2001) 10658 10667. [25] P.C. Ray, J. Leszczynski, Two-photon absorption and first nonlinear optical properties of ionic octupolar molecules: structure-function relationships and solvent effects, J. Phys. Chem. A 109 (30) (2005) 6689 6696. [26] W.T. Chen, U. Mahmood, R. Weissleder, C.H. Tung, Arthritis imaging using a near-infrared fluorescence folate-targeted probe, Arthrit. Res. Ther. 7 (2) (2005) R310 R317. [27] S. Santra, C. Kaittanis, J.M. Perez, Cytochrome c encapsulating theranostic nanoparticles: a novel bifunctional system for targeted delivery of therapeutic membrane-impermeable proteins to tumors and imaging of cancer therapy, Mol. Pharm. 7 (4) (2010) 1209 1222. [28] X.H. Gao, Y.Y. Cui, R.M. Levenson, L.W.K. Chung, S.M. Nie, In vivo cancer targeting and imaging with semiconductor quantum dots, Nat. Biotechnol. 22 (8) (2004) 969 976. [29] Y.M. Yang, Q. Shao, R.R. Deng, C. Wang, X. Teng, K. Cheng, et al., In vitro and in vivo uncaging and bioluminescence imaging by using photocaged upconversion nanoparticles, Angew. Chem. Int. Ed. 51 (13) (2012) 3125 3129. [30] H.B. Na, I.C. Song, T. Hyeon, Inorganic nanoparticles for MRI contrast agents, Adv. Mater. 21 (21) (2009) 2133 2148.

284

THERANOSTIC BIONANOMATERIALS

[31] Z. Jia, L. Song, F. Zang, J. Song, W. Zhang, C. Yan, et al., Active-target T1-weighted MR imaging of tiny hepatic tumor via RGD modified ultra-small Fe3O4 nanoprobes, Theranostics 6 (11) (2016) 1780 1791. [32] L. Cheng, C. Wang, X.X. Ma, Q.L. Wang, Y. Cheng, H. Wang, et al., Multifunctional upconversion nanoparticles for dual-modal imaging-guided stem cell therapy under remote magnetic control, Adv. Funct. Mater. 23 (3) (2013) 272 280. [33] K.M.L. Taylor, W.J. Rieter, W.B. Lin, Manganese-based nanoscale metal-organic frameworks for magnetic resonance imaging, J. Am. Chem. Soc. 130 (44) (2008) 14358 14359. [34] K. Brindle, New approaches for imaging tumour responses to treatment, Nat. Rev. Cancer 8 (2) (2008) 94 107. [35] H. Lusic, M.W. Grinstaff, X-ray-computed tomography contrast agents, Chem. Rev. 113 (3) (2013) 1641 1666. [36] D. Kim, S. Park, J.H. Lee, Y.Y. Jeong, S. Jon, Antibiofouling polymer-coated gold nanoparticles as a contrast agent for in vivo x-ray computed tomography imaging, J. Am. Chem. Soc. 129 (24) (2007) 7661 7665. [37] L. Wen, L. Chen, S. Zheng, J. Zeng, G. Duan, Y. Wang, et al., Ultrasmall biocompatible WO32x nanodots for multi-modality imaging and combined therapy of cancers, Adv. Mater. 28 (25) (2016) 5072 5079. [38] J. Zhou, M.X. Yu, Y. Sun, X.Z. Zhang, X.J. Zhu, Z.H. Wu, et al., Fluorine-18-labeled Gd3 1 /Yb3 1 /Er3 1 co-doped NaYF4 nanophosphors for multimodality PET/MR/UCL imaging, Biomaterials 32 (4) (2011) 1148 1156. [39] W. Shang, C. Zeng, Y. Du, H. Hui, X. Liang, C. Chi, et al., Core-shell gold nanorod@metal-organic framework nanoprobes for multimodality diagnosis of glioma, Adv. Mater. 29 (3) (2017) 1604381. [40] J. Wang, J. Liu, Y. Liu, L. Wang, M. Cao, Y. Ji, et al., Gd-hybridized plasmonic Au-nanocomposites enhanced tumor-interior drug permeability in multimodal imaging-guided therapy, Adv. Mater. 28 (40) (2016) 8950 8958. [41] E. Huynh, B.Y. Leung, B.L. Helfield, M. Shakiba, J.A. Gandier, C.S. Jin, et al., In situ conversion of porphyrin microbubbles to nanoparticles for multimodality imaging, Nat. Nanotechnol. 10 (4) (2015) 325 332. [42] K.H. Min, H.S. Min, H.J. Lee, D.J. Park, J.Y. Yhee, K. Kim, et al., pH-controlled gas-generating mineralized nanoparticles: a theranostic agent for ultrasound imaging and therapy of cancers, ACS Nano 9 (1) (2015) 134 145. [43] C. Pfister, G. Gravis, G. Pignot, A. Flechon, M. Soulie, C. Chevreau, Randomized phase III study of gemcitabine and cisplatin (GC) versus dose dense methotrexate, vinblastine, doxorubicin and cisplatin (DDMVAC) in the perioperative setting for patients with locally advanced transitional cell cancer of the bladder: The French GETUG/AFU V05 VESPER trial, J. Clin. Oncol. 36 (6) (2018) TPS530. [44] D.B. Longley, D.P. Harkin, P.G. Johnston, 5-Fluorouracil: mechanisms of action and clinical strategies, Nat. Rev. Cancer 3 (5) (2003) 330 338. [45] C. Bailly, Contemporary challenges in the design of topoisomerase II inhibitors for cancer chemotherapy, Chem. Rev. 112 (7) (2012) 3611 3640. [46] V. Shanmugam, S. Selvakumar, C.S. Yeh, Near-infrared light-responsive nanomaterials in cancer therapeutics, Chem. Soc. Rev. 43 (17) (2014) 6254 6287. [47] J.A. Webb, R. Bardhan, Emerging advances in nanomedicine with engineered gold nanostructures, Nanoscale 6 (5) (2014) 2502 2530. [48] M. Boyce, J. Yuan, Cellular response to endoplasmic reticulum stress: a matter of life or death, Cell Death Differ. 13 (3) (2006) 363 373. [49] Z. Zhang, J. Wang, C. Chen, Near-infrared light-mediated nanoplatforms for cancer thermochemotherapy and optical imaging, Adv. Mater. 25 (28) (2013) 3869 3880.

Chapter 11 • Two-Dimensional Nanomaterials in Cancer Theranostics

285

[50] L. Tong, Q.S. Wei, A. Wei, J.X. Cheng, Gold nanorods as contrast agents for biological imaging: optical properties, surface conjugation and photothermal effects, Photochem. Photobiol. 85 (1) (2009) 21 32. [51] N. Khlebtsov, L. Dykman, Biodistribution and toxicity of engineered gold nanoparticles: a review of in vitro and in vivo studies, Chem. Soc. Rev. 40 (3) (2011) 1647 1671. [52] X.J. Yang, X. Liu, Z. Liu, F. Pu, J.S. Ren, X.G. Qu, Near-infrared light-triggered, targeted drug delivery to cancer cells by aptamer gated nanovehicles, Adv. Mater. 24 (21) (2012) 2890 2895. [53] M.P. Melancon, M. Zhou, C. Li, Cancer theranostics with near-infrared light-activatable multimodal nanoparticles, Acc. Chem. Res. 44 (10) (2011) 947 956. [54] Y.N. Xia, W.Y. Li, C.M. Cobley, J.Y. Chen, X.H. Xia, Q. Zhang, et al., Gold nanocages: from synthesis to theranostic applications, Acc. Chem. Res. 44 (10) (2011) 914 924. [55] Z. Liu, A.C. Fan, K. Rakhra, S. Sherlock, A. Goodwin, X.Y. Chen, et al., Supramolecular stacking of doxorubicin on carbon nanotubes for in vivo cancer therapy, Angew. Chem. Int. Ed. 48 (41) (2009) 7668 7672. [56] G.M.F. Calixto, J. Bernegossi, L.M. de Freitas, C.R. Fontana, M. Chorilli, Nanotechnology-based drug delivery systems for photodynamic therapy of cancer: a review, Molecules 21 (3) (2016) 342. [57] N.L. Oleinick, R.L. Morris, T. Belichenko, The role of apoptosis in response to photodynamic therapy: what, where, why, and how, Photoch. Photobio. Sci. 1 (1) (2002) 1 21. [58] D.E.J.G.J. Dolmans, D. Fukumura, R.K. Jain, Photodynamic therapy for cancer, Nat. Rev. Cancer 3 (5) (2003) 380 387. [59] P. Agostinis, K. Berg, K.A. Cengel, T.H. Foster, A.W. Girotti, S.O. Gollnick, et al., Photodynamic therapy of cancer: an update, CA Cancer J. Clin. 61 (4) (2011) 250 281. [60] C.E. Dunbar, K.A. High, J.K. Joung, D.B. Kohn, K. Ozawa, M. Sadelain, Gene therapy comes of age, Science 359 (6372) (2018) 175. [61] T.B. Lentz, S.J. Gray, R.J. Samulski, Viral vectors for gene delivery to the central nervous system, Neurobiol. Dis. 48 (2) (2012) 179 188. [62] X.D. Yuan, S. Naguib, Z.Q. Wu, Recent advances of siRNA delivery by nanoparticles, Expert Opin. Drug Deliv. 8 (4) (2011) 521 536. [63] A.M. Scott, J.D. Wolchok, L.J. Old, Antibody therapy of cancer, Nat. Rev. Cancer 12 (4) (2012) 278 287. [64] M.J. Allen, V.C. Tung, R.B. Kaner, Honeycomb carbon: a review of graphene, Chem. Rev. 110 (1) (2010) 132 145. [65] B.A. Chen, M. Liu, L.M. Zhang, J. Huang, J.L. Yao, Z.J. Zhang, Polyethylenimine-functionalized graphene oxide as an efficient gene delivery vector, J. Mater. Chem. 21 (21) (2011) 7736 7741. [66] H.Q. Bao, Y.Z. Pan, Y. Ping, N.G. Sahoo, T.F. Wu, L. Li, et al., Chitosan-functionalized graphene oxide as a nanocarrier for drug and gene delivery, Small 7 (11) (2011) 1569 1578. [67] X.H. Liu, D.M. Ma, H. Tang, L. Tan, Q.J. Xie, Y.Y. Zhang, et al., Polyamidoamine dendrimer and oleic acid-functionalized graphene as biocompatible and efficient gene delivery vectors, ACS Appl. Mater. Interfaces 6 (11) (2014) 8173 8183. [68] Y.Z. Pan, H.Q. Bao, N.G. Sahoo, T.F. Wu, L. Li, Water-soluble poly(N-isopropylacrylamide)-graphene sheets synthesized via click chemistry for drug delivery, Adv. Funct. Mater. 21 (14) (2011) 2754 2763. [69] W. Zhang, Z.Y. Guo, D.Q. Huang, Z.M. Liu, X. Guo, H.Q. Zhong, Synergistic effect of chemophotothermal therapy using PEGylated graphene oxide, Biomaterials 32 (33) (2011) 8555 8561. [70] M. Li, X.J. Yang, J.S. Ren, K.G. Qu, X.G. Qu, Using graphene oxide high near-infrared absorbance for photothermal treatment of Alzheimer’s disease, Adv. Mater. 24 (13) (2012) 1722 1728. [71] X.Y. Chia, A.Y.S. Eng, A. Ambrosi, S.M. Tan, M. Pumera, Electrochemistry of nanostructured layered transition-metal dichalcogenides, Chem. Rev. 115 (21) (2015) 11941 11966.

286

THERANOSTIC BIONANOMATERIALS

[72] M. Chhowalla, H.S. Shin, G. Eda, L.J. Li, K.P. Loh, H. Zhang, The chemistry of two-dimensional layered transition metal dichalcogenide nanosheets, Nat. Chem. 5 (4) (2013) 263 275. [73] K. Kalantar-zadeh, J.Z. Ou, T. Daeneke, M.S. Strano, M. Pumera, S.L. Gras, Two-dimensional transition metal dichalcogenides in biosystems, Adv. Funct. Mater. 25 (32) (2015) 5086 5099. [74] Z.B. Li, S.L. Wong, Functionalization of 2D transition metal dichalcogenides for biomedical applications, Mater. Sci. Eng. C Mater. 70 (2017) 1095 1106. [75] T. Liu, S.X. Shi, C. Liang, S.D. Shen, L. Cheng, C. Wang, et al., Iron oxide decorated MoS2 nanosheets with double PEGylation for chelator-free radio labeling and multimodal imaging guided photothermal therapy, ACS Nano 9 (1) (2015) 950 960. [76] S.G. Wang, X. Li, Y. Chen, X.J. Cai, H.L. Yao, W. Gao, et al., A facile one-pot synthesis of a twodimensional MoS2/Bi2S3 composite theranostic nanosystem for multi-modality tumor imaging and therapy, Adv. Mater. 27 (17) (2015) 2775 2782. [77] J.Q. Chen, C.B. Liu, D.H. Hu, F. Wang, H.W. Wu, X.J. Gong, et al., Single-layer MoS2 nanosheets with amplified photoacoustic effect for highly sensitive photoacoustic imaging of orthotopic brain tumors, Adv. Funct. Mater. 26 (47) (2016) 8715 8725. [78] C.F. Zhang, Y. Yong, L. Song, X.H. Dong, X. Zhang, X.F. Liu, et al., Multifunctional WS2@poly(ethylene imine) nanoplatforms for imaging guided gene-photothermal synergistic therapy of cancer, Adv. Healthcare Mater. 5 (21) (2016) 2776 2787. [79] L. Cheng, A. Kamkaew, S. Shen, H.F. Valdovinos, H.Y. Sun, R. Hernandez, et al., Facile preparation of multifunctional WS2/WOx nanodots for chelator-free Zr-89-labeling and in vivo PET imaging, Small 12 (41) (2016) 5750 5758. [80] S.G. Wang, J.L. Zhao, H.L. Yang, C.Y. Wu, F. Hu, H.Z. Chang, et al., Bottom-up synthesis of WS2 nanosheets with synchronous surface modification for imaging guided tumor regression, Acta Biomater. 58 (2017) 442 454. [81] L. Cheng, C. Yuan, S.D. Shen, X. Yi, H. Gong, K. Yang, et al., Bottom-up synthesis of metal-ion-doped WS2 nanoflakes for cancer theranostics, ACS Nano 9 (11) (2015) 11090 11101. [82] J. Kim, H. Kim, W.J. Kim, Single-layered MoS2-PEI-PEG nanocomposite-mediated gene delivery controlled by photo and redox stimuli, Small 12 (9) (2016) 1184 1192. [83] J. Lee, J. Kim, W.J. Kim, Photothermally controllable cytosolic drug delivery based on core-shell MoS2porous silica nanoplates, Chem. Mater. 28 (17) (2016) 6417 6424. [84] W.Y. Yin, L. Yan, J. Yu, G. Tian, L.J. Zhou, X.P. Zheng, et al., High-throughput synthesis of single-layer MoS2 nanosheets as a near-infrared photothermal-triggered drug delivery for effective cancer therapy, ACS Nano 8 (7) (2014) 6922 6933. [85] T. Liu, C. Wang, W. Cui, H. Gong, C. Liang, X.Z. Shi, et al., Combined photothermal and photodynamic therapy delivered by PEGylated MoS2 nanosheets, Nanoscale 6 (19) (2014) 11219 11225. [86] S.S. Chou, B. Kaehr, J. Kim, B.M. Foley, M. De, P.E. Hopkins, et al., Chemically exfoliated MoS2 as nearinfrared photothermal agents, Angew. Chem. Int. Ed. 52 (15) (2013) 4160 4164. [87] Y. Yong, L.J. Zhou, Z.J. Gu, L. Yan, G. Tian, X.P. Zheng, et al., WS2 nanosheet as a new photosensitizer carrier for combined photodynamic and photothermal therapy of cancer cells, Nanoscale 6 (17) (2014) 10394 10403. [88] L.Y. Hao, H.J. Song, Y.Y. Su, Y. Lv, A cubic luminescent graphene oxide functionalized Zn-based metalorganic framework composite for fast and highly selective detection of Cu2 1 ions in aqueous solution, Analyst 139 (4) (2014) 764 770. [89] J. Park, Q. Jiang, D.W. Feng, H.C. Zhou, Controlled generation of singlet oxygen in living cells with tunable ratios of the photochromic switch in metal-organic frameworks, Angew. Chem. Int. Ed. 55 (25) (2016) 7188 7193.

Chapter 11 • Two-Dimensional Nanomaterials in Cancer Theranostics

287

[90] H.Y. Li, Y.L. Wei, X.Y. Dong, S.Q. Zang, T.C.W. Mak, Novel Tb-MOF embedded with viologen species for multi-photofunctionality: photochromism, photomodulated fluorescence, and luminescent pH sensing, Chem. Mater. 27 (4) (2015) 1327 1331. [91] M. Zhao, Y. Wang, Q. Ma, Y. Huang, X. Zhang, J. Ping, et al., Ultrathin 2D metal-organic framework nanosheets, Adv. Mater. 27 (45) (2015) 7372 7378. [92] Y.W. Zhao, J. Ling, S.G. Li, M. Li, A.R. Liu, S.Q. Liu, Synthesis of porphyrin-based two-dimensional metal-organic framework nanodisk with small size and few layers, J. Mater. Chem. A 6 (6) (2018) 2828 2833. [93] X.C. Gao, M.J. Zhai, W.H. Guan, J.J. Liu, Z.L. Liu, A. Damirin, Controllable synthesis of a smart multifunctional nanoscale metal-organic framework for magnetic resonance/optical imaging and targeted drug delivery, ACS Appl. Mater. Interfaces 9 (4) (2017) 3455 3462. [94] P. Horcajada, T. Chalati, C. Serre, B. Gillet, C. Sebrie, T. Baati, et al., Porous metal-organic-framework nanoscale carriers as a potential platform for drug delivery and imaging, Nat. Mater. 9 (2) (2010) 172 178. [95] D.M. Liu, C.B. He, C. Poon, W.B. Lin, Theranostic nanoscale coordination polymers for magnetic resonance imaging and bisphosphonate delivery, J. Mater. Chem. B 2 (46) (2014) 8249 8255. [96] T. He, B. Ni, S. Zhang, Y. Gong, H. Wang, L. Gu, et al., Ultrathin 2D zirconium metal-organic framework nanosheets: preparation and application in photocatalysis, Small 14 (16) (2018) e1703929. [97] X.D. Zhang, X. Xie, H. Wang, J.J. Zhang, B.C. Pan, Y. Xie, Enhanced photoresponsive ultrathin graphitic-phase C3N4 nanosheets for bioimaging, J. Am. Chem. Soc. 135 (1) (2013) 18 21. [98] X.D. Zhang, H.X. Wang, H. Wang, Q. Zhang, J.F. Xie, Y.P. Tian, et al., Single-layered graphitic-C3N4 quantum dots for two-photon fluorescence imaging of cellular nucleus, Adv. Mater. 26 (26) (2014) 4438 4443. [99] R. Chen, J.F. Zhang, Y. Wang, X.F. Chen, J.A. Zapien, C.S. Lee, Graphitic carbon nitride nanosheet@metal-organic framework core-shell nanoparticles for photo-chemo combination therapy, Nanoscale 7 (41) (2015) 17299 17305. [100] Y.Q. Dong, Q. Wang, H.S. Wu, Y.M. Chen, C.H. Lu, Y.W. Chi, et al., Graphitic carbon nitride materials: sensing, imaging and therapy, Small 12 (39) (2016) 5376 5393. [101] P. Yasaei, B. Kumar, T. Foroozan, C.H. Wang, M. Asadi, D. Tuschel, et al., High-quality black phosphorus atomic layers by liquid-phase exfoliation, Adv. Mater. 27 (11) (2015) 1887 1892. [102] C.X. Sun, L. Wen, J.F. Zeng, Y. Wang, Q. Sun, L.J. Deng, et al., One-pot solventless preparation of PEGylated black phosphorus nanoparticles for photoacoustic imaging and photothermal therapy of cancer, Biomaterials 91 (2016) 81 89. [103] H. Wang, X.Z. Yang, W. Shao, S.C. Chen, J.F. Xie, X.D. Zhang, et al., Ultrathin black phosphorus nanosheets for efficient singlet oxygen generation, J. Am. Chem. Soc. 137 (35) (2015) 11376 11382. [104] D. Yang, G.X. Yang, P.P. Yang, R.C. Lv, S.L. Gai, C.X. Li, et al., Assembly of Au plasmonic photothermal agent and iron oxide nanoparticles on ultrathin black phosphorus for targeted photothermal and photodynamic cancer therapy, Adv. Funct. Mater. 27 (18) (2017) 1700371. [105] H.U. Lee, S.Y. Park, S.C. Lee, S. Choi, S. Seo, H. Kim, et al., Black phosphorus (BP) nanodots for potential biomedical applications, Small 12 (2) (2016) 214 219. [106] W.S. Chen, J. Ouyang, H. Liu, M. Chen, K. Zeng, J.P. Sheng, et al., Black phosphorus nanosheet-based drug delivery system for synergistic photodynamic/photothermal/chemotherapy of cancer, Adv. Mater. 29 (5) (2017) 1603864. [107] M. Qiu, D. Wang, W.Y. Liang, L.P. Liu, Y. Zhang, X. Chen, et al., Novel concept of the smart NIR-lightcontrolled drug release of black phosphorus nanostructure for cancer therapy, Proc. Natl. Acad. Sci. U. S.A. 115 (3) (2018) 501 506.

288

THERANOSTIC BIONANOMATERIALS

[108] P. Gunawan, R. Xu, Direct control of drug release behavior from layered double hydroxides through particle interactions, J. Pharm. Sci. 97 (10) (2008) 4367 4378. [109] M.A. Thyveetil, P.V. Coveney, H.C. Greenwell, J.L. Suter, Computer simulation study of the structural stability and materials properties of DNA-intercalated layered double hydroxides, J. Am. Chem. Soc. 130 (14) (2008) 4742 4756. [110] L. Desigaux, M. Ben Belkacem, P. Richard, J. Cellier, P. Leone, L. Cario, et al., Self-assembly and characterization of layered double hydroxide/DNA hybrids, Nano Lett. 6 (2) (2006) 199 204. [111] L. Li, W.Y. Gu, J.Z. Chen, W.Y. Chen, Z.P. Xu, Co-delivery of siRNAs and anti-cancer drugs using layered double hydroxide nanoparticles, Biomaterials 35 (10) (2014) 3331 3339. [112] Y. Chen, D.L. Ye, M.Y. Wu, H.R. Chen, L.L. Zhang, J.L. Shi, et al., Break-up of two-dimensional MnO2 nanosheets promotes ultrasensitive pH-triggered theranostics of cancer, Adv. Mater. 26 (41) (2014) 7019 7026.

12 Polymeric Micelles for Tumor Theranostics Huaping Zhang, Peng Mi DE PARTMENT OF RADIOLOGY, CENTER FOR M EDICAL IMAGING, AND STATE KEY LABORA TORY OF BIOTHERAPY, WEST C HINA HOSPITAL, SICHUAN UNIVERSITY, COLLABORA TIVE INNOVATION CE NTER FOR B IOTHERAPY, CHENGDU, P.R. CHINA

12.1 Introduction Cancer is one of the biggest threats to public health, causing millions of deaths and an increasing number of new cases each year [1,2]. Accurate and early diagnosis of cancer is critically important for effective treatment, which could greatly improve people’s health and decrease the mortality rate. The detection of neoplastic lesions could provide pathological information on tumors, enabling monitoring of their development, as well as providing feedback on the therapeutic results. Molecular imaging has become an indispensible tool for detecting tumors as well as the biological processes within tumors [3] in preclinical studies and clinical trials [3,4]. Several molecular imaging modalities could be applied to acquire the anatomical or molecular information of organs and tissues, such as optical imaging, magnetic resonance imaging (MRI), ultrasound, positron emission tomography (PET), singlephoton emission computed tomography (CT) (SPECT), and X-ray CT. Compared to biopsy, molecular imaging provides a visible and physically noninvasive method for disease screening and positioning, as well as monitoring therapeutic effects in biological systems without surgical interference. The property of each molecular imaging modality has been summarized in Table 12 1, including the imaging quality, resolution, depth, quality, contrast agents (CAs), benefits, and limitations. CAs are generally required to improve the imaging quality and emit signals in biological systems to make them visible for detection [5]. Although a myriad of CAs have been applied in clinical applications, they are difficult to apply for cancer assessment, because they do not have tumor selectivity or targeting ability. In addition, rapid clearance of the low-molecular-weight CAs makes it difficult to achieve a diagnostic CA dose in tumors for sufficient contrast enhancement. Therefore drug-delivery systems, such as polymeric micelles [6], liposomes [7], inorganic nanoparticles [8], and polymersomes etc., [9] have been applied to specifically delivery CAs to tumors for molecular imaging with improved diagnostic selectivity and sensitivity.

Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00012-2 © 2019 Elsevier Inc. All rights reserved.

289

Table 12–1

Imaging Modalities for Cancer Imaging

Modality

Detection

Resolution Depth

Optical

Fluorescence

2 3 mm

MRI

Magnetic field 10 100 μm No limit

Ultrasound Ultrasonic waves

,1 cm

Quality Imaging Agents Low

High

50 μm

Several cm High

PET

γ-ray

1 2 mm

No limit

High

SPECT

γ-Ray

1 2 mm

No limit

High

CT

X-ray

50 μm

No limit

High

Benefits

Organic dyes, inorganic High sensitivity, noninvasive, multichannel, real-time nanoparticles, imaging, cheap, easy to graphene oxide handle Excellent spatial resolution, no Gd(III) chelates, iron penetration limitation, oxide nanoparticles, quantitative results, no manganese ionizing radiation, molecular imaging possible Noninvasive, real-time imaging, Microbubbles, cheap, easy handle nanobubbles, gas-generating nanoparticles 18 F-, 64Cu-, 11C-labeled High sensitivity and resolution, tracers limitless penetration depth, quantitative data 99m Tc-, 111In-labeled High sensitivity and resolution, tracers limitless penetration depth, no need of cyclotron Depicts anatomical features Bi-compounds, precisely iodinated compounds

Limitations Low spatial resolution, limited tissue penetration, autofluorescence Relatively expensive, low intensity, poor contrast, long acquisition time

Limited penetration, cannot penetrate bone or lung tissue

Expensive, ionizing radiation, low spatial resolution Relatively expensive, ionizing radiation, low spatial resolution, semi-quantitative Ionizing radiation, high dose of contrast agents, not definitive diagnostic results

CT, Computed tomography; MRI, magnetic resonance imaging; PET, positron emission tomography; SPECT, single-photon emission computed tomography.

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291

Among the drug-delivery systems, polymeric micelles that have self-assembled from amphiphilic block copolymers have attracted much attention for drug delivery [10 13] and cancer theranostics [14], while several candidates are under clinical trial [15,16]. Polymeric micelles are generally constructed to be 10 100 nm in diameter, and composited with a poly (ethylene glycol) (PEG) shell and a hydrophobic core (Fig. 12 1A). Polymeric micelles provide a platform for cancer therapy and diagnosis (theranostics), which could co-load nucleic

(A)

Cargoes

Shell

Chemotherapeutics Nucleic acids Proteins Peptides Imaging probes

Type Length Density Crosslinking Charge

Ligand Probe Drug Nucleic acid

Core

Targeting ligands

Hydrophobic Anionic Cationic Drug-conjugate Unimolecular structures Crosslinking π –π stacking

10–100 nm

Small molecules: sugar, carbohydrate, growth factors, vitamins, phenylboronic acid Biomacromolecules: antibodies, antibody fragment, aptamer, protein, peptide

(B) Ligand

Antitumor drug Contrast agent

Polymeric micelle Tumor tissue Nucleus

EPR effect Blood vessel

Lymphatic vessel

Impaired lymphatic drainage

FIGURE 12–1 Polymeric micelles for cancer theranostics. (A) The composition of polymeric micelles. (B) Polymeric micelles target tumor tissues for cancer theranostics. The polymeric micelles could be specifically accumulated in tumor tissues through the EPR effect for cancer diagnosis and therapy. EPR, Enhanced permeability and retention.

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acids, anticancer drugs, and antibodies inside the core for therapy, while incorporating CAs inside the core or conjugating on the surface for imaging. The PEG shell could protect the drugs inside the core from degradation by enzymes. Some targeting moieties, such as sugar, growth factors, phenylboronic acid, antibodies, and aptamer could be decorated on their surface to selectively interact with receptors that are highly expressed on tumor cells to increase the targeting ability. The polymeric micelles are stable in the blood circulation, and could deliver drugs/CAs to tumor tissues through the enhanced permeability and retention (EPR) effect (Fig. 12 1B) [17]. The targeting moieties of micelles could specifically interact with the epitopes that are overexpressed on cancer cells to increase the diagnostic selectivity and therapeutic limitations, and reduce their distribution in normal regions. Polymeric micelles with theranostic functions could be utilized for cancer diagnosis, image-guided therapy, tracing polymeric micelles in the body, and monitoring the therapeutic effects. In this chapter we focus on recent advances of polymeric micelles for tumor optical imaging, MRI, multifunctional imaging, image-guided therapy, and theranostics.

12.2 Polymeric Micelles for Tumor Imaging By loading imaging agents in polymeric micelles, it could be applied for tumor molecular imaging, as well as studying the biological manners of micelles, such as tracing their distribution/location in biological organisms, investigating their biological pathways, collecting pathological information, monitoring the therapeutic effects, and guiding cancer treatment. Here, polymeric micelles for optical imaging, MRI, and multifunctional/model imaging have been focused on.

12.2.1 Polymeric Micelles for Optical Imaging Optical imaging is the easiest way to investigate objects that probed by dyes in a real time manner. Compared to other imaging modalities, optical imaging has the advantages of abundant accessible choices of dyes, easy labeling, noninvasiveness, multichannel imaging function, as well as real-time imaging ability [18]. For in vivo optical imaging, it is required to conquer the poor tissue penetration and avoid background signal from the normal regions. Therefore polymeric micelles have been applied to increase the specificity and sensitivity for optical imaging [19 21]. The dyes could be conjugated to the polymers or physically trapped inside the core of polymeric micelles, which facilitates the applications of investigating polymeric micelles in biological organisms, such as location/microdistribution, biologic manners/pathway, as well as bioactivity. For instance, the dye-labeled polymeric micelles could be applied for tumor optical imaging. For in vivo imaging, it needs polymeric micelles to specifically probe tumors without signal at the background. The tumor microenvironment-responsive polymeric micelles that could specifically release the dyes in tumor tissues could be applied for tumor specific optical imaging. Recently, ultra pH-sensitive polymeric micelles loading with fluorescent dyes have been established for tumor detection [22]. There was no fluorescence in

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FIGURE 12–2 Schematic illustration of ultra pH-sensitive (UPS) polymeric micelles for tumor optical imaging. (A) The polymeric micelles can be activated in acidic tumor extracellular fluid with fluorescence “ON” for tumor imaging. (B) The polymeric micelles are very sensitive to the pH drop. (C) The pH-sensitive polymeric micelles for in vivo tumor imaging. (D) The pH-sensitive polymeric micelles installed with targeting moieties for highly selective in vivo tumor imaging. Adapted with permission from Y. Wang, et al., A nanoparticle-based strategy for the imaging of a broad range of tumours by nonlinear amplification of microenvironment signals, Nat. Mater. 13 (2014) 204 212.

blood circulation at normal pH (pH 7.4), but with strong fluorescence “ON” when responding to tumor pH (pH 6.5), demonstrating high potential for accurate tumor optical imaging (Fig. 12 2) [23]. Optical imaging provides the most convenient modality for biological imaging, especially for experimental and preclinical studies. However, the limited penetration of light (approximately 2 3 mm) makes it difficult to be applied for clinical tumor imaging, especially for detecting tumors inside the body. Thus, the imaging modalities with deep tissue penetration, such as MRI, have high potential for anatomical imaging of tumors in the clinic.

12.2.2 Polymeric Micelles for Tumor Magnetic Resonance Imaging MRI is a widely used molecular imaging modality in the clinic, which can be applied for anatomical imaging at high spatial resolution (1 mm) with soft-tissue imaging ability. It also has

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the function to evaluate metabolic changes in tissues. However, its application is highly limited by the disadvantages of low intensity/sensitivity and long signal acquisition time [24,25]. Therefore CAs have been designed to improve the imaging quality by enhancing the contrast of tissues for MRI, mainly including T1-weighted, T2-weighted, and chemical exchange saturation transfer (CEST) CAs. Among them, the T1-weighted CAs are preferred by doctors because the positive contrast enhancement is more reliable and easier for interpretation than the negative contrast enhancement from T2-weighted CAs [26,27]. However, current clinical available T1-weighted CAs mainly are Gd(III) chelates, which have intrinsic low molecular relaxivity (r1), very little contrast enhancement, and without tumor-targeting ability. Thus, CAs that could specifically probe tumors is required. Polymeric micelles have been applied to deliver MRI CAs to tumors and then specifically enhance the contrast of tumor tissues for precise diagnosis [28]. For instance, the clinically used MRI CAs, such as Gd-DTPA, could be incorporated into polymeric micelles to target tumors through the EPR effect for tumor diagnosis. The Gd-DTPA-loaded polymeric micelles have demonstrated significantly higher relaxivity than Gd-DTPA [29,30], due to the confinement of CAs inside the core of polymeric micelles, leading to higher contrast enhancement in tumors than Gd-DTPA. In addition, the T2-weighted CAs of superparamagnetic iron-oxide nanoparticles (SPIONs) also could be loaded inside polymeric micelles to exhibit negative contrast enhancement in tumors with increased diagnostic sensitivity [6,31,32]. Also, by installing specific ligands on the surface of polymeric micelles, it could increase the diagnostic selectivity by specifically interacting with the epitopes on tumor cells [33 36]. Polymeric micelles also could be designed to respond to pathological parameters in the tumor microenvironment, such as low pH [37,38], redox potential [39,40], enzymes [41 43], and hyaluronidase [44,45], to specially enhance the contrast, and even amplify and convert the pathological signals to visible contrast enhancement. For instance, tumor pH-activatable calcium phosphate (CaP) polymeric micelles incorporating CAs of Mn21 have been reported, which could amplify the contrast of tumor regions when the Mn21 was released and interacted with environmental proteins [46]. The polymeric micelles could specifically and sensitively enhance the contrast of tumors, indicating tumor hypoxia and liver micrometastasis (Fig. 12 3), which are critical important for detecting tumor malignancy, as tumor hypoxia is related to drug resistance and metastasis accounts for 90% of tumor deaths. In addition, this study provides a strategy to amplify the contrast in tumor regions for diagnosis, which could be applied to accurate diagnosis of small tumors.

12.2.3 Polymeric Micelles for Tumor Multimodality Imaging Although many imaging modalities could be applied for tumor imaging, each imaging modality has its intrinsic advantages and limitations, such as sensitivity and spatial resolution [47 49], which could be solved by combining two or more imaging modalities to improve the diagnostic accuracy and efficiency. Thus, CAs that are suitable for multimodality imaging are required, and polymeric micelles provide a suitable platform to load different types of CAs to facilitate multiple imaging modalities. For instance, multifunctional polymeric

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FIGURE 12–3 Noninvasive MR imaging of tumor malignancy by pH-activatable polymeric micelles with a signal amplification function. (A) Schematic illustration of the pH-activatable polymeric micelles (PEGMnCaP). (B) 3D MR imaging of solid tumors with contrast enhancement by PEGMnCaP. (C) MR imaging of tumor hypoxic regions contrasted by PEGMnCaP. The regions of hypoxia in tumor tissues were confirmed by pimonidazole staining, while the nuclei were stained with hematoxylin. (D) MR imaging of C26 tumors after administration of PEGMnCaP, with CSI of lactate finally obtained, confirming the hypoxic regions are overlapping with high-lactate regions. (E) MR imaging of liver metastasis contrasted by PEGMnCaP, showing specific contrast enhancement in liver micrometastatic regions. 3D, Three-dimensional; CSI, chemical shift imaging; MR, magnetic resonance. Adapted with permission from P. Mi, et al., A pH-activatable nanoparticle with signal-amplification capabilities for noninvasive imaging of tumour malignancy, Nat. Nanotechnol. 11 (2016) 724 730.

micelles incorporating photosensitizer chlorine e6 (Ce6), Gd31-based chelating agents and near-infrared (NIR) dye of IR825, could offer contrast for three different imaging modalities, including optical imaging, MR imaging, and photoacoustic tomography (PAT) (Fig. 12 4) [50]. In addition, polymeric micelles loaded with other CAs, such as radionuclide rhenium188 (188Re) together with NIR dye of IR-780 iodide, could be applied for dual optical and micro single-photon emission computed tomography (SCPET) [20]. By loading with desired CAs, polymeric micelles could be applied for multimodal imaging by optical-MRI [51], optical-CT [52], optical-SCPET [53], PET-MRI [54], SCPET-MRI [55], etc., demonstrating high

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FIGURE 12–4 (A) Schematic illustration of polymeric micelles for multimodal tumor imaging. Ce6 is anchored on the backbone of C18PMH-PEG polymer via a short PEG linker. Gd31 forms a chelate complex with Ce6. IR825, a water-insoluble NIR dye, is then encapsulated inside the formed polymeric micelles. (B) In vivo multimodal tumor imaging. (a) In vivo fluorescence and (b) MR and (c) in vivo photoacoustic imaging of 4T1 tumor-bearing mice taken at different time points post i.v. injection of the polymeric micelles. Dashed circles in (a) and (b) highlight the tumor, while arrows in (b) point to the heart. NIR, Near-infrared. Adapted with permission from H. Gong, et al., Engineering of multifunctional nano-micelles for combined photothermal and photodynamic therapy under the guidance of multimodal imaging, Adv. Funct. Mater. 24 (2014) 6492 6502.

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performance for tumor precise imaging. Although great efforts have been made to develop polymeric micelles for multimodal molecular imaging, their translation to clinical applications is still incomplete, because the systems are difficult to satisfy each imaging modality.

12.2.4 Polymeric Micelles for Tumor Theranostics A myriad of bioactive compounds, such as anticancer drugs, nucleic acids, and antibodies could also be loaded in polymeric micelles together with CAs for cancer theranostics. The CAs in the theranostic micelles could be applied for tumor imaging, image-guided therapy, tracing the therapeutics in the body, as well as monitoring the therapeutic effects, while the bioactive compounds could be applied for tumor treatment. Therefore it provides multiple functions for cancer diagnosis, therapy, therapy management, and drug design validation based on a single system [16,56,57]. The theranostic micelles also could be functionalized to specifically increase the diagnostic selectivity by targeting the tumor sites and then releasing cargos there when responding to specific stimuli in tumor microenvironments, such as pH, redox, and enzymes, etc. [14,19,53,58,59]. For instance, multifunctional polymeric micelles loaded with built-in pH-tunable “on off” triggers could respond to an intracellular pH drop to release the anticancer drug paclitaxel (PTX), while surface-coated folate could actively target tumors and incorporated quantum dots could be applied for optical imaging [60]. Theranostic micelles also could provide anatomical information for image-guided therapy, which can avoid potential interference with normal regions. For example, we recently developed gadolinium (Gd) chelate-loaded polymeric micelles for MRI-guided gadolinium neutron capture therapy (GdNCT) of solid tumors (Fig. 12 5) [61]. The polymeric micelles were constructed using PEG-b-poly(aspartic acid) to hybridize with CaP for particle formulation and size control, while incorporating the clinically available MRI CAs of Gd-diethylenetriaminepentaacetic acid (Gd-DTPA) inside the CaP core (Fig. 12 5A). The Gd-DTPA-loaded polymeric micelles could deliver Gd-DTPA to tumors to detect the location and edge of tumors by MRI (Fig. 12 5B). Thereafter, the tumor tissues were irradiated by thermal neutrons guided by MR images. In tumor tissues, the Gd atoms could capture thermal neutrons to emit γ rays after the nuclear reaction to kill cancer cells and eradicate tumors (Fig. 12 5C and D), while the thermal neutron irradiation is not harmful to normal regions without the distribution of Gd. This study provides a strategy for detecting tumors by molecular imaging modality to provide pathological information for tumor radiotherapy, while avoiding unnecessary irradiation to normal organ/tissue. In addition, polymeric micelles incorporating Gd-based MRI CAs and platinum (Pt) anticancer drugs through reversible metal chelation of Pt could be applied for imaging and chemotherapy of intractable human pancreatic tumors [62]. The imaging function of the micelles could be applied to noninvasively monitor therapeutic outcomes of the platinum anticancer drug. In addition, a series of “all-in-one” theranostic micelles has been developed with multiple imaging functions, which also could load several drugs to achieve synergistic therapeutic effects for cancer treatment [19,20,50,58,63 65]. However, it is difficult to clarify the pharmacokinetics and conduct pharmacological studies for clinical translation.

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FIGURE 12–5 In vivo MR imaging-guided gadolinium neutron capture therapy of tumors with Gd-DTPA-loaded polymeric micelles. (A) Scheme of Gd-DTPA/CaP hybrid micelles targeting tumors for gadolinium neutron capture therapy (GdNCT). In vivo images of mice bearing subcutaneous C26 tumors after intravenous injection of Gd-DTPA/ CaP (B) and Gd-DTPA (C), respectively. (D) The growth curve of the subcutaneous C26 tumors demonstrates the therapeutic effects of Gd-DTPA/CaP. GdNCT, Gadolinium neutron capture therapy; MR, magnetic resonance. Adapted with permission from P. Mi, et al., Hybrid calcium phosphate-polymeric micelles incorporating gadolinium chelates for imaging-guided gadolinium neutron capture tumor therapy. ACS Nano 9 (2015) 5913 5921.

12.3 Conclusions and Perspective Much effort has been engaged in developing polymeric micelles for cancer treatment, and several candidates are under clinical trial, forecasting promising future clinical applications. Polymeric micelles loaded with both CAs and therapeutic compounds have attracted much attention for imaging-guided therapy and tumor theranostics, demonstrating several advantages for cancer treatment. However, the clinical translation of polymeric micelles is still limited to several candidates, while it is still paving the way for clinical translation of theranostic polymeric micelles. The slow clinical translation of the theranostic polymeric micelles is

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mainly due to the hurdles of formulation, pharmacological study, and the fact that it is difficult to match the best effects of each bioactive compound and imaging agent. It is difficult to precisely control the doses of imaging agents and anticancer compounds inside one system, while a minimum amount of imaging agents is required for tumor visualization and a high therapeutic dose of bioactive compounds is required for cancer treatment. Thus, it is better to find a more suitable method for good formulation of the theranostic polymeric micelles with the desired amount of imaging agents and anticancer compounds. Moreover, by incorporating two or more compounds into one system, each component may have different pharmacological properties, which should be carefully considered when designing the theranostic polymeric micelles. In addition, the opposing timelines and different locations of the imaging agents and therapeutic compounds should be considered, as the imaging agents are expected to reach the target tissue in a short time for diagnosis/detection, while it generally takes a long time for the therapeutic compounds to reach the tumors. Therefore, for developing theranostic polymeric micelles for clinical translation, future efforts may be expected to integrate reliable targeting moieties, approved therapeutic compounds, and imaging agents into one polymeric micelle system to specifically target tumor regions for tumor diagnosis, and then release the therapeutic compounds for effective therapy.

Acknowledgments This work was supported by the National Key R&D Program of China (2017YFA0207900).

References [1] S. McGuire, World Cancer Report 2014. Geneva, Switzerland: World Health Organization, International Agency for Research on Cancer, WHO Press, 2015, Adv. Nutr. 7 (2016) 418 419. [2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2016, CA Cancer J. Clin. 66 (2016) 7 30. [3] R. Weissleder, M.J. Pittet, Imaging in the era of molecular oncology, Nature 452 (2008) 580 589. [4] R. Weissleder, Molecular imaging in cancer, Science 312 (2006) 1168 1171. [5] E.C. Cho, C. Glaus, J. Chen, M.J. Welch, Y. Xia, Inorganic nanoparticle-based contrast agents for molecular imaging, Trends Mol. Med. 16 (2010) 561 573. [6] H. Ai, et al., Magnetite-loaded polymeric micelles as ultrasensitive magnetic-resonance probes, Adv. Mater. 17 (2005) 1949 1952. [7] G. Mikhaylov, et al., Ferri-liposomes as an MRI-visible drug-delivery system for targeting tumours and their microenvironment, Nat. Nanotechnol. 6 (2011) 594 602. [8] G. Lin, P. Mi, C. Chu, J. Zhang, G. Liu, Inorganic nanocarriers overcoming multidrug resistance for cancer theranostics, Adv. Sci. 3 (2016) 1600134 1601600. [9] Z. Li, et al., Soft nanomaterial-based targeting polymersomes for near-infrared fluorescence multispectral in vivo imaging, Nanoscale 4 (2012) 7097 7105. [10] K. Sakai-Kato, et al., General considerations regarding the in vitro and in vivo properties of block copolymer micelle products and their evaluation, J. Controlled Release 210 (2015) 76 83. [11] K. Osada, R.J. Christie, K. Kataoka, Polymeric micelles from poly(ethylene glycol)-poly(amino acid) block copolymer for drug and gene delivery, J. R. Soc., Interface 6 (Suppl. 3) (2009) S325 S339.

300

THERANOSTIC BIONANOMATERIALS

[12] Y. Kakizawa, K. Kataoka, Block copolymer micelles for delivery of gene and related compounds, Adv. Drug Deliv. Rev. 54 (2002) 203 222. [13] K. Kataoka, A. Harada, Y. Nagasaki, Block copolymer micelles for drug delivery: design, characterization and biological significance, Adv. Drug Deliv. Rev. 47 (2001) 113 131. [14] H. Cabral, N. Nishiyama, K. Kataoka, Supramolecular nanodevices: from design validation to theranostic nanomedicine, Acc. Chem. Res. 44 (2011) 999 1008. [15] H. Cabral, K. Kataoka, Progress of drug-loaded polymeric micelles into clinical studies, J. Controlled Release 190 (2014) 465 476. [16] P. Mi, F. Wang, N. Nishiyama, H. Cabral, Molecular cancer imaging with polymeric nanoassemblies: from tumor detection to theranostics, Macromol. Biosci. (2016). Available from: https://doi.org/10.1002/ mabi.201600305. [17] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Res. 46 (1986) 6387 6392. [18] J. Wang, et al., Imaging-guided delivery of RNAi for anticancer treatment, Adv. Drug Deliv. Rev. 104 (2016) 44 60. [19] C.K. Huang, C.L. Lo, H.H. Chen, G.H. Hsiue, Multifunctional micelles for cancer cell targeting, distribution imaging, and anticancer drug delivery, Adv. Funct. Mater. 17 (2007) 2291 2297. [20] C.L. Peng, et al., Multimodal image-guided photothermal therapy mediated by 188Re-labeled micelles containing a cyanine-type photosensitizer, ACS Nano 5 (2011) 5594 5607. [21] Y. Li, et al., A smart and versatile theranostic nanomedicine platform based on nanoporphyrin, Nat. Commun. 5 (2014) 4712. [22] M. Sun, et al., Fluorescent supramolecular micelles for imaging-guided cancer therapy, Nanoscale 8 (2016) 5302 5312. [23] Y. Wang, et al., A nanoparticle-based strategy for the imaging of a broad range of tumours by nonlinear amplification of microenvironment signals, Nat. Mater. 13 (2014) 204 212. [24] C. Khemtong, C.W. Kessinger, J. Gao, Polymeric nanomedicine for cancer MR imaging and drug delivery, Chem. Commun. (2009) 3497 3510. [25] H. Kobayashi, M.R. Longmire, M. Ogawa, P.L. Choyke, S. Kawamoto, Multiplexed imaging in cancer diagnosis: applications and future advances, Lancet Oncol. 11 (2010) 589 595. [26] E. Terreno, D.D. Castelli, A. Viale, S. Aime, Challenges for molecular magnetic resonance imaging, Chem. Rev. 110 (2010) 3019 3042. [27] R. Weissleder, M. Nahrendorf, M.J. Pittet, Imaging macrophages with nanoparticles, Nat. Mater. 13 (2014) 125 138. [28] K. Shiraishi, K. Kawano, T. Minowa, Y. Maitani, M. Yokoyama, Preparation and in vivo imaging of PEGpoly(L-lysine)-based polymeric micelle MRI contrast agents, J. Controlled Release 136 (2009) 14 20. [29] P. Mi, et al., Gd-DTPA-loaded polymer-metal complex micelles with high relaxivity for MR cancer imaging, Biomaterials 34 (2013) 492 500. [30] P. Mi, et al., Hydrothermally synthesized PEGylated calcium phosphate nanoparticles incorporating GdDTPA for contrast enhanced MRI diagnosis of solid tumors, J. Controlled Release 174 (2014) 63 71. [31] H.Y. Yoon, et al., Hydrotropic magnetic micelles for combined magnetic resonance imaging and cancer therapy, J. Controlled Release 160 (2012) 692 698. [32] M. Kumagai, et al., Enhanced magnetic resonance imaging of experimental pancreatic tumor in vivo by block copolymer-coated magnetite nanoparticles with TGF-beta inhibitor, J. Controlled Release 140 (2009) 306 311.

Chapter 12 • Polymeric Micelles for Tumor Theranostics

301

[33] C. Khemtong, et al., In vivo off-resonance saturation magnetic resonance imaging of alphavbeta3targeted superparamagnetic nanoparticles, Cancer Res. 69 (2009) 1651 1658. [34] N. Nasongkla, et al., Multifunctional polymeric micelles as cancer-targeted, MRI-ultrasensitive drug delivery systems, Nano Lett. 6 (2006) 2427 2430. [35] N. Nasongkla, et al., cRGD-functionalized polymer micelles for targeted doxorubicin delivery, Angew. Chem. Int. Ed. 43 (2004) 6323 6327. [36] O. Veiseh, J.W. Gunn, M. Zhang, Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging, Adv. Drug Deliv. Rev. 62 (2010) 284 304. [37] X.N. Huang, et al., Multi-chromatic pH-activatable F-19-MRI nanoprobes with binary ON/OFF pH transitions and chemical-shift barcodes, Angew. Chem. Int. Ed. 52 (2013) 8074 8078. [38] G.H. Gao, et al., Magnetite-nanoparticle-encapsulated pH-responsive polymeric micelle as an MRI probe for detecting acidic pathologic areas, Small 6 (2010) 1201 1204. [39] P. Kuppusamy, et al., Noninvasive imaging of tumor redox status and its modification by tissue glutathione levels, Cancer Res. 62 (2002) 307 312. [40] S. Aime, M. Botta, E. Gianolio, E. Terreno, A p(O-2)-responsive MRI contrast agent based on the redox switch of manganese(II/III) porphyrin complexes, Angew. Chem. Int. Ed. 39 (2000) 747 750. [41] C.E. Callmann, et al., Therapeutic enzyme-responsive nanoparticles for targeted delivery and accumulation in tumors, Adv. Mater. 27 (2015) 4611 4615. [42] C.M. Overall, O. Kleifeld, Tumour microenvironment opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy, Nat. Rev. Cancer 6 (2006) 227 239. [43] E.S. Olson, et al., Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases, Proc. Natl. Acad. Sci. U.S.A. 107 (2010) 4311 4316. [44] A.L. Harris, Hypoxia—a key regulatory factor in tumour growth, Nat. Rev. Cancer 2 (2002) 38 47. [45] W.R. Wilson, M.P. Hay, Targeting hypoxia in cancer therapy, Nat. Rev. Cancer 11 (2011) 393 410. [46] P. Mi, et al., A pH-activatable nanoparticle with signal-amplification capabilities for non-invasive imaging of tumour malignancy, Nat. Nanotechnol. 11 (2016) 724 730. [47] S.M. Janib, A.S. Moses, J.A. MacKay, Imaging and drug delivery using theranostic nanoparticles, Adv. Drug Deliv. Rev 62 (2010) 1052 1063. [48] A. Louie, Multimodality imaging probes: design and challenges, Chem. Rev. 110 (2010) 3146 3195. [49] D.E. Lee, et al., Multifunctional nanoparticles for multimodal imaging and theragnosis, Chem. Soc. Rev. 41 (2012) 2656 2672. [50] H. Gong, et al., Engineering of multifunctional nano-micelles for combined photothermal and photodynamic therapy under the guidance of multimodal imaging, Adv. Funct. Mater. 24 (2014) 6492 6502. [51] R. Kumar, et al., Combined magnetic resonance and optical imaging of head and neck tumor xenografts using Gadolinium-labelled phosphorescent polymeric nanomicelles, Head Neck Oncol. 2 (2010) 35. [52] J. Zhang, et al., In vivo tumor-targeted dual-modal fluorescence/CT imaging using a nanoprobe coloaded with an aggregation-induced emission dye and gold nanoparticles, Biomaterials 42 (2015) 103 111. [53] R. Zhang, et al., Peptide-conjugated polymeric micellar nanoparticles for Dual SPECT and optical imaging of EphB4 receptors in prostate cancer xenografts, Biomaterials 32 (2011) 5872 5879. [54] L.W. Starmans, et al., 89 Zr- and Fe-labeled polymeric micelles for dual modality PET and T1-weighted MR imaging, Adv. Healthc. Mater. 4 (2015) 2137 2145. [55] Y. Miura, et al., Polymeric micelle platform for multimodal tomographic imaging to detect scirrhous gastric cancer, ACS Biomater. Sci. Eng. 1 (2015) 1067 1076.

302

THERANOSTIC BIONANOMATERIALS

[56] J. Kim, Y. Piao, T. Hyeon, Multifunctional nanostructured materials for multimodal imaging, and simultaneous imaging and therapy, Chem. Soc. Rev. 38 (2009) 372 390. [57] K. Park, et al., New generation of multifunctional nanoparticles for cancer imaging and therapy, Adv. Funct. Mater. 19 (2009) 1553 1566. [58] W. Li, et al., Smart hyaluronidase-actived theranostic micelles for dual-modal imaging guided photodynamic therapy, Biomaterials 101 (2016) 10 19. [59] H.C. Tsai, et al., Graft and diblock copolymer multifunctional micelles for cancer chemotherapy and imaging, Biomaterials 31 (2010) 2293 2301. [60] W. Wang, D. Cheng, F. Gong, X. Miao, X. Shuai, Design of multifunctional micelle for tumor-targeted intracellular drug release and fluorescent imaging, Adv. Mater. 24 (2012) 115 120. [61] P. Mi, et al., Hybrid calcium phosphate-polymeric micelles incorporating gadolinium chelates for imaging-guided gadolinium neutron capture tumor therapy, ACS Nano 9 (2015) 5913 5921. [62] S. Kaida, et al., Visible drug delivery by supramolecular nanocarriers directing to single-platformed diagnosis and therapy of pancreatic tumor model, Cancer Res. 70 (2010) 7031 7041. [63] A. Yuan, et al., Self-assembled PEG-IR-780-C13 micelle as a targeting, safe and highly-effective photothermal agent for in vivo imaging and cancer therapy, Biomaterials 51 (2015) 184 193. [64] X. Liu, et al., Photosensitizer cross-linked nano-micelle platform for multimodal imaging guided synergistic photothermal/photodynamic therapy, Nanoscale 8 (2016) 15323 15339. [65] H. Zhang, X. Kong, Y. Tang, W. Lin, Hydrogen sulfide triggered charge-reversal micelles for cancertargeted drug delivery and imaging, ACS Appl. Mater. Interfaces 8 (2016) 16227 16239.

13 Theranostic Biomaterials for Regulation of the Blood Brain Barrier Rui Pedro Moura1, Flávia Sousa1,2,3,4, Andreia Almeida2,3,4, Soraia Pinto2,3, Bruno Sarmento1,2,3 1 CESPU—INSTITUTE O F INV ESTIGATION A ND ADVANCED FORMATION IN HEALTH SCIENCES AND TECHNOLOGIES, GANDRA, PORTUGAL 2 i3S—INSTITUTE OF INVESTIGATION AND INNOVATION IN HEALTH, UNIVERISITY OF PORTO, PORTO, PORTUGAL 3 INEB —NATIONAL INSTITUTE OF B IOME DICAL E NGINEERING , UNIVERS ITY OF P ORTO, P ORTO, PORTUGAL 4 I C B AS —A BE L S A L A ZA R ' S IN S TIT UT E O F B I O ME D I CA L S C I E NC E S , UN I V ER S I T Y OF PORTO, PORT O, PORTUGAL

Abbreviations RGD BBB BBTB CNS GDNF GLUT-1 HIV iPSCs MRI MMP-9 NSCs PEG PET ROS SPIONs TJs TNF-α VEGF

arginylglycylaspartic acid blood brain barrier blood brain tumor barrier central nervous system glial-derived neurotrophic factor glucose transporter-1 human immunodeficiency virus induced pluripotent stem cells magnetic resonance imaging matrix metalloproteinase-9 neural stem cells polyethylene glycol positron emission tomography reactive oxygen species superparamagnetic iron-oxide nanoparticles tight junctions tumor necrosis factor alpha vasoendothelial growth factor

13.1 Introduction The blood brain barrier (BBB) is a highly specialized barrier that was demonstrated for the first time by Paul Ehlrich, in 1885 [1]. It acts as the main protector of the central nervous system (CNS), transiently limiting the passage of many compounds [2]. It also ensures that Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00013-4 © 2019 Elsevier Inc. All rights reserved.

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the CNS gets a constant and controlled influx of nutrients and controls how inflammatoryresponsive cells react to disruptions of the normal BBB environment [3]. It is composed of specialized endothelial cells that differ from other endothelial cells in the human body (e.g., vein endothelial cells) due to the high expression of tight junctions (TJs) between them, the lack of fenestrations, and reduced pinocytic activity [4,5]. The TJs confer to the endothelial cells the extremely limiting behavior that is observed at the BBB, due to unique features when compared to other TJs expressed on the body, such as their P-face/E-face ratio. The TJs are composed of occludins and claudins, two types of integral membrane proteins. Dysregulation regarding these proteins, such as changes to their phosphorylation, has been directly linked to certain CNS pathologies [6]. Therefore it is absolutely required that the TJs at the BBB hold all their natural features without abnormal alterations in order to safeguard the CNS [7]. In addition to endothelial cells, the BBB is also composed of astrocyte end-feet, pericytes, perivascular macrophages, microglia, neurons, and a basement membrane, which is composed mainly of extracellular matrix proteins and the main target when considering the repair and/or regeneration of a damaged BBB [8,9]. These components, together, have earned the denomination of the “neurovascular unit,” to demonstrate their coordinated teamwork to ensure the safety and regulation of the CNS. Also, the BBB phenotype and role is not only achieved and maintained by the special endothelial cells, but by adequate and carefully regulated messages by all these cellular “players.” Both astrocytes and pericytes are constantly regulating BBB features to ensure that no imbalances occur in the CNS, such as controlling TJ permeability or controlling the expression of nutrient transporters [8,10]. CNS diseases are some of the most problematic diseases to fight against, mainly due to the previously mentioned BBB innate limitation to the delivery of agents [11]. However, in plenty of pathological processes of the CNS, there is a distinct dysfunction in the BBB protective role, which can range from transient BBB disruption, such as disruption of the effectiveness of TJs or a decrease in P-glycoprotein efflux transporters [12,13], to deficiencies in certain nutrients, such as deficiency in glucose due to downregulation of the glucose transporter-1 (GLUT-1) [14]. Therefore it is of the utmost necessity to not only combat the diseases pharmacologically, but to attempt to promote the regeneration of the BBB to fully restore the CNS’s natural behavior and ensure that it is, once again, protected by its trustworthy barrier. Scaffolds or carbon nanotubes here seem to have an emerging role when it comes to BBB regeneration. Their potential stems from their ability to act as substrates to promote the growth of axons and the extracellular matrix of the brain, but also from the ability to incorporate within them stem cells to further increase the differentiation process and regeneration of damaged BBB areas [15,16]. Furthermore, diagnosing and/or providing vital imaging of the BBB, for instance through the use of gold nanoparticles to monitor BBB permeability, can also serve an important role to help in guiding the treatment and to obtain more efficient results, without increasing the toxicity of the treatment [17]. Thus, nanoscale biomaterials present fantastic opportunities to achieve this goal due to their overall safety and the ability to modulate several of their features to ensure an adequate and satisfying treatment [18]. Nanobiomaterials that can accomplish both a diagnostic purpose coupled with a therapeutic action, earn themselves the definition of a theranostic

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agent [19]. The idea to develop theranostic agents arose due to the mixed results in treatments for certain diseases. It was thought that coupling a diagnostic agent with a therapeutic action could ameliorate these results. Although commonly applied for cancers, these theranostic agents can, and should, have other applications [20]. Therefore the rationale is that the use of these theranostic agents can provide a massive help when dealing with complications related to the BBB, allowing the actions taken to be thorough and controlled. This review aims to summarize the current biomaterials that are extensively used in both diagnostic and therapeutic applications at the BBB, as well as presenting and discussing biomaterials that can combine both of these actions, acting as theranostic biomaterials.

13.2 The Blood Brain Barrier 13.2.1 The Blood Brain Barrier’s Structure and Function The CNS requires an extremely delicate balance on its intake of nutrients, cells, and fluids constantly, to ensure the brain performs its tasks adequately without imbalances that can cause brain damage. To accomplish this, the CNS is isolated from the remainder of the body by an extremely limiting and well-regulated barrier, the BBB [21]. The BBB is composed mostly of a special type of endothelial cell, avoiding the transport of substances through the BBB to the CNS. The TJs between specialized endothelial cells limit the paracellular transport occurring at the BBB. Low rates of transcytosis at these cells further represent the low permeation observed at the BBB [22,23]. It is well known that there is higher transendothelial electrical resistance at the BBB when compared to other endothelial tissues of the body [24]. Lastly, the BBB has a high expression of efflux transporters (e.g., P-glycoprotein) and multidrug resistance proteins [25]. Due to these features, only very specific substances and nutrients can routinely cross the BBB, and to do so, specific transport pathways are exploited. These pathways include: [8,26,27] 1. Paracellular pathway through the TJs of the endothelial cells, however, due to the high effectiveness of these TJs this pathway is seldom used and only exploited by very small hydrophilic agents; 2. Diffusion through the cell membranes (unique to gas molecules and extremely small and lipophilic agents); 3. Exploiting transport proteins existent at the surface of the endothelial cells (the GLUT-1 transporter and the transport of glucose); 4. Cell-surface receptor-mediated transcytosis, where a molecule interacts with certain receptors expressed by endothelial cells of the BBB and is, subsequently, interiorized by the endothelial cells (transferrin receptor, insulin receptor); 5. Adsorptive transcytosis, that differs from receptor-mediated transcytosis as no receptor is needed to internalize the compound, it is internalized through surface charge interactions (albumin, other plasma proteins).

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Joining the endothelial cells, the BBB is also composed of astrocytes, pericytes, perivascular macrophages, microglia, and neurons. Astrocytes and pericytes are responsible for the maintenance of certain functions of the BBB, such as its permeability, through TJ modulation, receptor and transporter expression, endothelial cell configuration, and electrolyte and fluid levels [8,28,29]. Macrophages are immune system cells which are mostly responsible for the immune response in certain CNS diseases, with their role thoroughly associated with certain degenerations and other diseases [30]. Microglia are also resident immune cells at the BBB, which become activated when the brain suffers a particular injury or when exposed to certain stimuli from the immune system. The particular microglia activation has been linked to transient BBB disruption in certain diseases, and can progress to neurodegenerations, so it is of the utmost importance that these cells are tightly regulated. Microglia can also modulate the BBB permeability, control immune-cell trafficking at the BBB and regulate angiogenesis [31,32]. Lastly, the basement membrane promotes the anchoring of all the previously mentioned cellular components of the BBB, contributing to the dynamic equilibrium observed [33]. It is made up of extracellular matrix compounds, which promote its structural integrity (i.e., collagen, elastin, laminin, fibronectin) and proteoglycans (i.e., perlecan and agrin) [34]. A correct association of the basement membrane is key to a healthy BBB, as if certain compounds are either missing or in low expression, there can be repercussions in the overall state of the BBB. To exemplify, an unhealthy basement membrane can result in weaker TJs and a weaker association between astrocyte end-feet and endothelial cells [35]. A brief description of the BBB and all the remaining components that make up the neurovascular unit can be seen in Fig. 13 1.

13.2.2 Role of the Blood Brain Barrier in Central Nervous System Diseases A functional CNS might be affected by many diseases, such as tumors (glioblastomas), neurodegenerative diseases (multiple sclerosis, Alzheimer’s disease), traumatic injuries (brain trauma), or vascular disorders (stroke). Recent advances have shown that most of these diseases promote a weakening or disruption of the BBB, coupled with inflammation of the CNS. Logically, when the brain’s main source of protection and regulation is damaged or hindered, the brain’s homeostasis will suffer. Thus, the combination of these processes is a negative factor for subsequent neurodegeneration, that can happen in some diseases when the brain’s protection is limited [36]. One of the most common CNS diseases is brain tumors, such as glioblastoma. Brain tumors can promote vascular growth within them, generating blood vessels that are different to those outside of the tumor. This led to the concept of a blood brain tumor barrier (BBTB) [37]. The BBTB is a “new” barrier that brain tumors can cause, with alterations to the BBB vasculature, leading to an abnormally permeable BBB. The formation of the BBTB has been linked with the tumor’s metabolic demands, which promotes an increase in

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FIGURE 13–1 Representative scheme of the BBB. The pericytes are located in the basement membrane, whilst the astrocytes extend their end-feet toward the endothelial cells, but are not in direct contact with them. The communication between these cells is mainly attributed to soluble factors [8]. The constant communication between all these cell “players” is what counts toward a healthy BBB. Pericytes and astrocytes are firmly anchored in the basement membrane of the BBB, a type of extracellular matrix mainly composed of proteins and proteoglycans. The extracellular matrix is also the main target when promoting repair and/or regeneration of the BBB due to its role in promoting the general equilibrium between the BBB and the brain. BBB, Blood brain barrier.

vasoendothelial growth factor (VEGF), leading to new blood vessel development. This effectively creates a new barrier that therapeutic agents must cross, to reach the tumor [38]. In multiple sclerosis, researchers have shown that there is a transient breakdown of the BBB due to proinflammatory cytokines, such as tumor necrosis factor alpha or certain interleukins, and activated immune cells (leukocytes) freely crossing the BBB in abnormally elevated rates. This can lead to reduced BBB organization, but also a decrease in laminin expression, a structural component of the basement membrane of the BBB [39,40]. These chemical mediators can hold a direct action in BBB endothelial cells, that actually modulates how leukocytes interact with the endothelial cells, further increasing their migration across the BBB. This abnormal amount of leukocytes can, and will, damage the BBB through the production of reactive oxygen species (ROS), explaining how multiple sclerosis progresses with BBB disruption [41]. In Alzheimer’s disease, it has been described that there are modified levels of agrin and other proteoglycans of the basement membrane of the BBB and, also, a reduced efficacy in the efflux mechanism of the BBB [42]. Similarly, in other neurodegenerative diseases, such as Parkinson’s disease and Huntington’s disease, this reduced effectiveness of P-glycoprotein is also reported [43]. Another way in which certain disorders can promote BBB disruption is through mechanisms that stimulate the expression of matrix metalloproteinases, a type of enzyme

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responsible for degrading certain extracellular matrix compounds. Examples of this BBB disruption mechanism can be found in stroke [44] and human immunodeficiency virus infections that have spread to the CNS, where the main target seems to be laminin [45]. Also, in both traumatic brain injury and an ischemic stroke event, at the core of the lesion, it is extremely common to note that the BBB can be fully damaged, resulting in its temporary (although long) absence. This will, eventually, lead to the accumulation and diffusion of molecules or compounds that were previously filtered by the BBB, creating certain gradients that can cause physiological imbalances and, actually promote the CNS to be more prone to other neurological diseases, such as Alzheimer’s or Parkinson’s diseases [46]. Traumatic brain injury can be characterized by not only neural cell death but also transient BBB breakdown, which promotes the activation of reactive glial cells, forming a glial scar to replace the necrotic tissue at the site of the injury [47]. At the beginning, the glial scar can be considered beneficial, as it will effectively separate the healthy from the damaged/dead portions of the brain and aid in the regeneration of the BBB. Also, it will eventually develop a limiting barrier that will prevent a harmonious regeneration of neural tissue [48]. Thus, there is evidence that plenty of CNS disorders, or injuries, can affect the BBB in certain ways. Therefore it is important to consider the BBB not only as a barrier limiting the CNS, but also as a promising therapeutical target when considering CNS diseases or injuries. Promoting the regeneration of the BBB and/or repairing the basement membrane and combating the underlying cause of BBB breakdown can prove to be a valuable asset when dealing with CNS pathologies and can increase the survivability of patients and improve their quality of life. Although they have not been explored to their full potential, biomaterials such as hydrogel scaffolds are excellent candidates to tackle this necessity.

13.3 Explaining Theranostic Agents—A Growing Concept Nanomaterials can be synthesized by a variety of materials and can be functionalized in a plethora of different ways, allowing a vast range of applications. The functionalization of nanomaterials might allow the targeting of specific receptors, increasing the targeting of a specific organ [49]. Thus, recent advances in nanotechnology and specifically in nanomedicine have seen an increase in agents that have earned the denomination of “theranostic agents.” This is attributed to the intrinsic features that nanomaterials have, such as unique imaging properties, an adequate size that allows full-body distribution, and the ability to functionalize their surface to prolong their half-life [50]. A theranostic agent can be defined as an agent which can combine both imaging/diagnostic properties with a therapeutic function [19,51]. Therefore this combination might bring a wide array of advantages to clinicians and researchers, such as the ability to promote imaging during a treatment regimen, without having to either perform it after application or before starting the treatment regimen. Another advantage of the use of nanostructures as theranostic agents comes from the fact that many nanostructures can already be effectively used as imaging agents, requiring only certain modifications, such as its content and surface functionalization to suit as therapeutic

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agents [20]. Some of these theranostic agents can be coupled with an external magnetic stimulus to allow increased directioning of the delivery route toward the CNS (e.g., iron-oxide nanoparticles [20] or microbubbles coupled with ultrasound promoting in situ transformations into nanoparticles, that will retain their cargo and their imaging properties) [52]. An example of this can be seen on the work carried out by Lammers et al., where poly(butyl cyanoacrylate) microbubbles carrying ultrasmall superparamagnetic iron-oxide nanoparticles (SPIONs) were used to enduce, and monitor, BBB permeability [53]. Thus, these theranostic agents can promote plenty of advantages when targeting the BBB as a treatment option for CNS disorders as their imaging properties can eventually demonstrate possible “leaky” sites in the BBB or sites where BBB function is impaired and immediately aim to repair or regenerate it. To sum, a nano-scaled theranostic agent should optimally hold the following features [20]: (1) imaging properties, to promote a better understanding of the treatment necessity and allow a more thorough approach; (2) possess diagnostic properties, to aid in the understanding of the underlying condition and the problems required to fix; (3) therapeutical action, to treat the underlying condition; and (4) safe, tolerable, and adequate for human use, preferentially avoiding nefarious side effects.

13.4 Nanobiomaterials Used to Repair and/or Regenerate the Blood Brain Barrier A healthy and fully functioning BBB is key to maintaining the CNS at peak efficiency and safeguarded from all threats. When the BBB is disrupted, due to the initiation of a certain disorder, or due to trauma, the tight regulation of nutrients and immune cells is effectively lost. What this can mean for the CNS is that, when a neurodegenerative process initiates, the BBB will no longer be a viable defense mechanism for the CNS. Therefore it is important to act upon the BBB, to prevent the degenerative process to overwhelm the CNS, up to a point where promoting the regeneration of the BBB no longer translates as an effective treatment option [54]. Therefore CNS disorders and BBB breakdown or disruption, are of the utmost interest to promote a fast and adequate regeneration or reparation of the extracellular matrix of the CNS to once again regain homeostasis in the CNS. Here, certain biomaterials have been employed to promote this, such as electrospun or hydrogel scaffolds, and carbon nanotubes. These biomaterials, transiently coupled with imaging materials, presented later in this review, make up the theranostic biomaterials that are effectively employed to “guide-and-fix” the BBB. The main goal aimed for, when using a specific repairing or regenerating biomaterial within the BBB, is to promote support for the neighboring cells so that regeneration can occur. Therefore it should be able to mimic the physiological interactions that normally occur at the site, such as nutrient transport, cell growth, and cell differentiation, without disturbing normal cell cell communication, and these biomaterials should interact with the resident cells at the BBB in a harmonious way to ensure no complications [55,56].

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13.4.1 Scaffolds A biomaterial scaffold is meant to act as a medium for cell growth and regeneration. They can be degradable or nondegradable, produced in several forms, and constituted by different compounds, depending on the objective and the necessity for the use of the scaffold [57]. Scaffolds should mimic the extracellular matrix of the BBB, therefore they can control the cells surrounding them and allow the exchange of nutrients and other needed compounds at the BBB. Scaffolds should also be able to allow the permeation of axons and other cells through their structure to ensure development of a perfect environment [58,59]. In addition, scaffolds are meant to prevent the appearance of inflammatory and immune responses and glial scars at the place of implantation, having moderate water composition and an affinity for water-based solutions to ease their inclusion in the organism [60]. Therefore, to sum, a scaffold must effectively act as an extracellular matrix, where the natural extracellular matrix has sustained damage or cannot accomplish its role appropriately, with the ultimate goal of promoting the regeneration, in this case, of the BBB. Scaffolds can be developed through different methods. Hydrogels are three-dimensional polymeric compounds that are stitched together through crosslinks, that can be either physical or chemical. Hydrogels have the innate ability, when in contact with water, to acquire a gel-like shape. These scaffolds can also show a certain antiinflammatory action, when their composition is similar to the constituents that surround the hydrogel [61]. They can be made up of natural extracellular matrix components, such as hyaluronic acid, chitosan, alginate, or of synthetic derivatives, such as polyethylene glycol (PEG) [59]. Another production method to obtain scaffolds is through electrospinning. This procedure can produce nano-scaled fibers with high porosity, promoting an adequate resemblance to the features of both laminin and collage hold within the extracellular matrix [59]. Lastly, scaffolds can also be elaborated through self-assembly. This is accomplished through hydrophobic interactions, where certain compounds can assemble into nanofiber scaffolds. These hold advantages when compared to electrospun nanofibers, as these will mimic the extracellular matrix more adequately due to the smaller fiber diameters and porous constitution [62]. Scaffolds can also be combined with certain vasoactive agents, growth factors, drugs, or therapeutic proteins to further promote development and regeneration of the injured areas of the brain, or coupled with extracellular matrix proteins, that are normally intrinsic to the BBB, to increase their ability to promote cell-adhesion and regeneration of the extracellular matrix and, subsequently, the BBB [63 65]. A work elaborated by Zhang et al. demonstrated that the use of gelatin-based scaffolds coupled with VEGF promotes the development of endothelial cells as well as astrocytes and microglia at the borders of the implanted scaffold, suggesting a possible regeneration of the BBB in areas where traumatic brain injury occurred [15]. Another study, conducted by Nga et al., highlighted the use of polycaprolactone-based scaffolds to promote the regeneration of the BBB after penetration in the brain injury. This study showed that these scaffolds promoted endothelial cell regeneration, did not promote further inflammation at the barrier, and contributed to a decrease in BBB permeation, which contributed to a reduction of

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cerebral edema [66]. Tian et al. showed the use of a hydrogel combining hyaluronic acid and poly-D-lysine (to promote cell adhesion) was effectively compatible with brain tissue and promoted the regeneration and the interaction between glial cells and the scaffold [67]. Similarly, Hou et al. showed that hyaluronic acid hydrogels coupled with laminin promoted cellular infiltration and repealed the glial scar formation [68]. Also, Cui et al. used hyaluronic acid scaffolds coupled with arginylglycylaspartic acid peptides and it was shown that hyaluronic acid scaffolds promoted axonal regeneration and cellular reorganization in injured portions of the brain [69]. Tate et al. thoroughly described the conditions of using methylcellulose-based scaffolds in traumatic brain injury conditions, highlighting that the scaffold did not exacerbate the injury or promote toxic responses [70]. However, the use of scaffolds to repair the architecture of the extracellular matrix of the BBB is not the only desirable effect they hold. Scaffolds can also promote the delivery of neural stem cells (NSCs) to promote cellular differentiation and regeneration at the BBB, without losing any of the functions a scaffold has when used to fix the broken-down architecture at the BBB. When exploiting scaffolds as delivery agents of these stem cells, the role of extracellular matrix proteins appears to be extremely beneficial, as it can promote their differentiation and integration at the site of transplantation [65]. The work carried out by Cheng et al. showed the benefit of using a self-assembling hydrogel coupled with NSCs, added with laminin derivatives to promote higher efficiency and efficacy to the role NSCs are meant to carry out at the CNS, managing to promote brain-injury repair and a repeal of glial scar formation [71]. Lam et al. reported the use of a hyaluronic acid hydrogel, coupled with induced pluripotent stem cells and NSCs in a stroke-injury in vivo study, showing that the hydrogel was able to induce the cells carried within to differentiate accordingly and promote the restoration of the damaged CNS, and, by extension, the BBB [72]. On the other hand, Zhong et al. employed hydrogels produced by thiol groups crosslinked with hyaluronan heparin collagen PEG-carrying neural progenitor cells in a stroke cavity. The main highlights of this work were a reduction in activated microglia and macrophages in the area, corresponding to an attenuation of the postinjury environment and the promotion of regeneration of the extracellular matrix [73]. Wang et al. used fibrous poly(ε-caprolactone) scaffolds coupled with glial cell line-derived neurotrophic factor-carrying NSCs to compare with the same NSCs but without the scaffold, highlighting the increased cellular differentiation and survivability of the NSCs when delivered coupled with the scaffold [74]. Regarding inorganic scaffolds, there is little-to-no information regarding their use repairing the BBB, possibly due to the toxicity these inorganic compounds could hold when in contact with CNS compounds.

13.4.2 Carbon Nanotubes Carbon nanotubes can be synthesized through two different methods: single-walled nanotubes, which consist of an individual layer of carbon entities in a tubular form; and multiwalled nanotubes, consisting of multiple layers of carbon molecules. Carbon nanotubes have emerged recently as biomaterial candidates to repair and/or regenerate the BBB due to

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their ability to adequately foster the development of NSCs cultured with them and their overall structural integrity and electrical conductivity [75]. Jan et al. showed through their work that NSCs when coupled with carbon nanotubes could be successfully differentiated in neurons, astrocytes, or oligodendrocytes, and that these promoted neuronal regeneration [76]. McKenzie et al. found that carbon-based biomaterials had the ability to repeal astrocytes, and therefore the formation of the glial scar that normally occurs when the BBB is damaged [77]. With that information in mind, researchers started to expand the possible role that carbon nanotubes could hold within the CNS and their ability to regenerate both CNS and the BBB. The work carried out by Galvan-Garcia et al. showed that carbon nanotubes promise to promote cellular attachment, growth and differentiation, and neuron survival and neurite development [78]. Indeed, all these qualities make carbon nanotubes an extremely appealing candidate when considering the BBB as a possible therapeutic target. Thus, using scaffolds or carbon nanotubes seems to hold promise when tackling CNS injuries, neurodegenerative diseases, or other complications that affect the BBB. The use of NSCs or other types of stem cells is also an extremely viable strategy. However, it is required to guarantee that these cells can effectively grow and differentiate. Although scaffolds or carbon nanotubes show promise and satisfying results both in vitro and in vivo, either alone or coupled with stem cells, nothing has yet been translated to human research and clinical trials.

13.5 Nanobiomaterials Used as Imaging and Diagnosing Agents at the Blood Brain Barrier For an agent to be considered a theranostic agent, as stated previously, it should not only have a therapeutic action at the BBB, but also a diagnostic role. Some biomaterials, mainly derivatives of metallic nanoparticles, are commonly employed to aid with imaging techniques, due to their excellent X-ray absorption coupled with relatively low toxicity [79]. Thus, after these nanoparticles have been administered, they are usually observed through magnetic resonance imaging (MRI) or positron emission tomography (PET). The main objective of imaging agents regarding the BBB in CNS disorders and/or injuries, is to visualize areas where the BBB was damaged, resulting in abnormal leaky areas, to correctly diagnose what the underlying cause for the leaky BBB is, and to eventually visualize the damage to act accordingly. These imaging biomaterials can also be employed to keep track of certain BBB disruption techniques in combination with treatment regimens [80].

13.5.1 Gold Nanoparticles Gold nanoparticles consist of a gold core with surface ligands usually attached. They are considered relatively stable and have shown low toxicity levels when employed in animal models [17]. Their general application as imaging agents is due to their relatively low level of interaction with physiological molecules or targets. Gold nanoparticles can also be employed to diagnose abnormal leaky BBB. Unlike some of the other metallic nanoparticles, gold

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nanoparticles have been shown to be relatively safe when targeting the CNS, further highlighting the possible role of gold nanoparticles in diagnosing abnormally elevated permeability in the BBB [17]. Although there are limited studies employing these nanoparticles in pathological situations, there are studies reporting the use of gold nanoparticles to determine BBB permeability. The work carried out by Frigell et al. employed gold glyconanoparticles, labeled with gadolinium-68 for PET detection, coupled with surface functionalization to promote higher CNS uptake, to determine BBB permeability [81]. This can be interesting to employ in pathological conditions, as, if the BBB is damaged, the permeability values will be higher. Sawyer et al. proposed a method, also using gold nanoparticles, to evaluate the BBB state during a foreign body response [82]. The study proposes a method where researchers could evaluate BBB integrity through the use of fluorescent gold nanoparticles and, afterwards, transmission electronic microscopy was used to detect the gold nanoparticles at the brain parenchyma of the animal model.

13.5.2 Quantum Dots Quantum dots are semiconducting fluorescent nanocrystals that have been employed recently in imaging and diagnostic roles due to their unique properties. The main property that gives quantum dots such a prominent role in imaging and diagnostic techniques is their ability to be observed under fluorescence microscopy, whilst maintaining their fluorescent properties for significantly longer periods of time than most fluorescent agents. However, optimization is still required when using quantum dots due to the possibility of these to aggregate within cells, potentially damaging them [83]. Some other features that make quantum dots interesting are their chemical stability and the ability to resist photobleaching [84]. The work carried out by Bonoiu et al. expertly demonstrated the theranostic properties quantum dots can hold within the BBB [85]. Quantum dots were complexed with matrixdegrading metalloproteinase-9 (MMP-9) small interfering RNA (siRNA) to promote the downregulation of the previously mentioned MMP, to attempt to preserve BBB integrity. After delivery, the fluorescent properties of quantum dots supplied data regarding BBB integrity, and the global expression of proteins of the extracellular matrix of the BBB was restored to normal values. This work adequately exemplifies the potential that a theranostic agent can hold both in imaging/diagnostic and therapeutic properties, when fighting CNS disorders with transient BBB breakdown.

13.5.3 Superparamagnetic Iron Oxide Nanoparticles SPIONs are nanoparticles with an iron-oxide derivative core, such as magnetite (Fe3O4), maghemite (γ-Fe2O3), or hermatite (α-Fe2O3) surrounded by an external hydrophilic coating [86]. Their size, shape, and external coating are the main features to take into account when preparing SPIONs in order to be directed at specific purposes. Their surface coating is of extreme interest when directing SPIONs at the BBB, as without the support of normal BBBcrossing aiding agents, such as lactoferrin, SPIONs will not adequately cross the BBB [87].

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The most interesting properties these nanoparticles hold, as the name implies, is their superparamagnetism. This allows SPIONs to be guided by an external magnetic stimulus, to promote their accumulation into a proper tissue. SPIONs have been studied as targeted MRI contrast agents, thus showing why SPIONs can be considered a powerful tool when dealing with faulty BBB. When the external magnetic stimulus is removed, SPIONs lose practically all their magnetic properties and do not aggregate as often, allowing them to evade the mononuclear phagocytic system for longer periods of time [86,88]. Similarly, PEGylated SPIONs are another way to avoid their aggregation and to increase their time in the circulation, due to the repulsion provided by PEG [89]. Both features contribute to an increase in the effectiveness of their imaging properties. In 1992, in one of the first studies regarding BBB imaging, Bulte et al. demonstrated that dextran-magnetite nanoparticles could be used to detect lesions or disruptions within the BBB. This was performed in a rat model where the rat’s cortex was purposely injured, in order to validate the ability to image BBB disruption [90]. The results showed that these SPIONs could adequately be used to monitor BBB disruption coupled with MRI. Liu et al. demonstrated in mice after postischemic damage that PEGylated SPIONs can adequately monitor BBB alterations and permeability values for up to 24 h with a single administration of the nanoparticles [91]. SPIONs were then observed through MRI, and the subsequent imaging data allowed researchers to understand the areas of the BBB where permeability was affected. Despite the many advantages these nanobiomaterials used in BBB imaging/diagnosis can provide, special focus should be paid to their potential toxicity. The formation of ROS, altering the natural function of resident microglia at the BBB, and autophagy dysfunctions have all been linked to metallic nanoparticles used in imaging features. Thus, special precautions have to be considered when engineering nanobiomaterials with the purpose of BBB imaging [92].

13.6 Conclusion and Future Perspectives CNS disorders and/or injuries are always a cause for concern due to the limitations of the possible treatment options. However, these injuries or disorders should not only be viewed as nefarious to the CNS but also toward the BBB. The BBB, as the CNS’s most efficient line of defense, should also be considered as a therapeutic option. Indeed, in a plethora of CNS disorders, there is notorious damage to the BBB, and this will increase the damage toward the CNS, as its main line of defense has been rendered inefficient. With the advancements in nanotechnology, and the increase in the usage of theranostic agents, targeting the BBB as a therapeutic option has become a prominent approach to limiting the progression of CNS disorders. The ability to employ an agent that has both the capabilities of localizing leaky or abnormally set portions of the BBB and combining it with a potential therapeutic agent can undoubtedly increase the rate of success when combating CNS disorders. Although there are few clinical data on these treatment options in humans,

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the results are promising. Current research goals should be aimed toward perfecting these techniques, and creating biomaterials that show no toxicity in humans.

Acknowledgments Flávia Sousa and Andreia Almeida would like to thank to FCT for funding the PhD scholarship (SFRH/BD/ 112201/2015 and SFRH/BD/118721/2016). This chapter is a result of project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was also financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274).

References [1] D. Ribatti, et al., Development of the blood brain barrier: a historical point of view, Anat. Rec. B New Anat. 289 (1) (2006) 3 8. [2] L.L. Rubin, J.M. Staddon, The cell biology of the blood-brain barrier, Annu. Rev. Neursci. 22 (1999) 11 28. [3] Y. Persidsky, et al., Blood brain barrier: structural components and function under physiologic and pathologic conditions, J. Neuroimmune. Pharmacol. 1 (3) (2006) 223 236. [4] B.W. Chow, C. Gu, The molecular constituents of the blood brain barrier, Trends Neurosci. 38 (10) (2015) 598 608. [5] P. Ballabh, A. Braun, M. Nedergaard, The blood brain barrier: an overview: structure, regulation, and clinical implications, Neurobiol. Dis. 16 (1) (2004) 1 13. [6] B. Nico, D. Ribatti, Morphofunctional aspects of the blood brain barrier, Curr. Drug. Metab. 13 (1) (2012) 50 60. [7] H. Wolburg, A. Lippoldt, Tight junctions of the blood brain barrier: development, composition and regulation, Vascul. Pharmacol. 38 (6) (2002) 323 337. [8] N.J. Abbot, L. Rönnbäck, E. Hansson, Astrocyte-endothelial interactions at the blood brain barrier, Nat. Rev. Neurosci. 7 (1) (2006) 41 53. [9] P.D. Yurchenco, B.L. Patton, Developmental and pathogenic mechanisms of basement membrane assembly, Curr. Pharm. Des. 15 (2009) 1277 1294. [10] C. Iadecola, Neurovascular regulation in the normal brain and in Alzheimer’s disease, Nat. Rev. Neurosci. 5 (5) (2004) 347 360. [11] N.J. Abbot, et al., Structure and function of the blood brain barrier, Neurobiol. Dis. 37 (1) (2010) 13 25. [12] S. McQuaid, et al., The effects of blood brain barrier disruption on glial cell function in multiple sclerosis, Biochem. Soc. Trans. 37 (Pt 1) (2009) 329 331. [13] L.M. Tai, et al., P-glycoprotein and breast cancer resistance protein restrict apical-to-basolateral permeability of human brainendothelium to amyloid-β, J. Cereb. Blood Flow Metab. 29 (6) (2009) 1079 1083. [14] R.N. Kalaria, The blood brain barrier and cerebrovascular pathology in Alzheimer’s disease, Ann. N.Y. Acad. Sci. 893 (1999) 113 125.

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[15] H. Zhang, et al., Gelatin Siloxane hybrid scaffolds with vascular endothelial growth factor induces brain tissue regeneration, Curr. Neurovasc. Res. 5 (2) (2008) 112 117. [16] O.A. Carballo-Molina, I. Velasco, Hydrogels as scaffolds and delivery systems to enhance axonal regeneration after injuries, Front Cell Neurosci. 9 (2015) 13. [17] D. Male, R. Gromnicova, C. McQuaid, Gold nanoparticles for imaging and drug transport to the CNS, Int. Rev. Neurobiol. 130 (2016) 155 198. [18] A.V. Singh, et al., Theranostic implications of nanotechnology in multiple sclerosis: a future perspective, Autoimmune Dis. 2012 (2012) 12. [19] S.M. Janib, A.S. Moses, J.A. MacKay, Imaging and drug delivery using theranostic nanoparticles, Adv. Drug. Deliv. Rev. 62 (11) (2010) 1052 1063. [20] J. Xie, S. Lee, X. Chen, Nanoparticle-based theranostic agents, Adv. Drug. Deliv. Rev. 62 (11) (2010) 1064 1079. [21] Y. Serlin, et al., Anatomy and physiology of the blood-brain barrier, Semin. Cell Dev. Biol. 38 (2015) 2 6. [22] R. Daneman, A. Prat, The blood brain barrier, Cold Spring Harb. Perspect. Biol. 7 (1) (2015) a020412. [23] B.I. Coomber, P.A. Stewart, Morphometric analysis of CNS microvascular endothelium, Microvasc. Res. 30 (1985) 99 115. [24] S.M. Stamatovic, R.F. Keep, A.V. Andjelkovic, Brain endothelial cell cell junctions: how to “open” the blood brain barrier, Curr. Neuropharmacol. 6 (3) (2008) 179 192. [25] A. Mahringer, G. Fricker, ABC transporters at the blood brain barrier, Expert Opin. Drug Metab. Toxicol. 12 (5) (2016) 499 508. [26] M.P. Pinto, et al., Targeted brain delivery nanoparticles for malignant gliomas, Nanomedicine (Lond.) 12 (1) (2017) 59 72. [27] J.L. Mikitsh, A.-M. Chacko, Pathways for small molecule delivery to the central nervous system across the blood-brain barrier, Perspect. Med. Chem. 6 (2014) 11 24. [28] A. Armulik, et al., Pericytes regulate the blood-brain barrier, Nature 468 (7323) (2010) 557 561. [29] R. Moura, A. Almeida, B. Sarmento, The role of non-endothelial cells on the penetration of nanoparticles through the blood brain barrier, Prog. Neurobiol. 159 (2017) 39 49. [30] G. Stollg, S. Jander, The role of microglia and macrophages in the pathophysiology of the CNS, Prog. Neurobiol. 58 (3) (1999) 233 247. [31] A. da Fonseca, et al., The impact of microglial activation on blood brain barrier in brain diseases, Front Cell Neurosci. 8 (2014) 362. [32] N. Dudvarski Stankovic, et al., Microglia-blood vessel interactions: a double-edged sword in brain pathologies, Acta Neuropathol. 131 (3) (2016) 347 363. [33] B. Obermeier, A. Verma, R.M. Ransohoff, The blood brain barrier, Handb. Clin. Neurol. 133 (2016) 39 59. [34] M. Thomsen, L. Routhe, T. Moos, The vascular basement membrane in the healthy and pathological brain, J. Cereb. Blood Flow Metab. 37 (10) (2017) 3300 3317. [35] M.J. Menezes, et al., Extracellular matrix protein laminin α2 regulates the maturation and function of the blood brain barrier, J. Neurosci. 34 (46) (2014) 15260 15280. [36] A.M. Palmer, The role of the blood brain barrier in neurodegenerative disorders and their treatment, J. Alzheimers Dis. 24 (4) (2011) 643 656. [37] J. Blakeley, Drug delivery to brain tumors, Curr. Neurol. Neurosci. Rep. 8 (3) (2008) 235 241. [38] O. van Tellingen, et al., Overcoming the blood brain tumor barrier for effective glioblastoma treatment, Drug Resist. Updat. 19 (2015) 1 12.

Chapter 13 • Theranostic Biomaterials for Regulation of the Blood Brain Barrier 317

[39] T. Oki, et al., Increased ability of peripheral blood lymphocytes to degrade laminin in multiple sclerosis, J. Neurol. Sci. 222 (1-2) (2004) 7 11. [40] A. Minagar, J.S. Alexander, Blood brain barrier disruption in multiple sclerosis, Mutl. Scler. 9 (6) (2003) 540 549. [41] C. Larochelle, J. Alvarez, A. Prat, How do immune cells overcome the blood brain barrier in multiple sclerosis? FEBS Lett. 585 (23) (2011) 3770 3780. [42] T. Berzin, et al., Agrin and microvascular damage in Alzheimer’s disease, Neurobiol. Aging 21 (2) (2000) 349 355. [43] A.L. Bartels, Blood brain barrier P-glycoprotein function in neurodegenerative disease, Curr. Pharm. Des. 17 (26) (2011) 2771 2777. [44] E.H. Lo, T. Dalkara, M.A. Moskowitz, Mechanisms, challenges and opportunities in stroke, Nat. Rev. Neurosci. 4 (5) (2003) 399 415. [45] K.R. Spindler, T.-H. Hsu, Viral disruption of the blood brain barrier, Trends Microbiol. 20 (6) (2012) 282 290. [46] J.E. Burda, M.V. Sofroniew, Reactive gliosis and the multicellular response to CNS damage and disease, Neuron 81 (2) (2014) 229 248. [47] G. Yiu, Z. He, Glial inhibition of CNS axon regeneration, Nat. Rev. Neurosci. 7 (2006) 617 627. [48] J. Silver, J. Miller, Regeneration beyond the glial scar, Nat. Rev. Neurosci. 5 (2004) 146 156. [49] C. Saraiva, et al., Nanoparticle-mediated brain drug delivery: overcoming blood brain barrier to treat neurodegenerative diseases, J. Controlled Release 235 (2016) 34 47. [50] T. Kim, S. Lee, X. Chen, Nanotheranostics for personalized medicine, Expert Rev. Mol. Diagn 13 (3) (2013) 257 269. [51] V.H. Tam, et al., Nanomedicine as a non-invasive strategy for drug delivery across the blood brain barrier, Int. J. Pharm. 515 (1-2) (2016) 331 342. [52] E. Huynh, et al., In situ conversion of porphyrin microbubbles to nanoparticles for multimodality imaging, Nat. Nanotechnol. 10 (4) (2015) 325 332. [53] T. Lammers, et al., Theranostic USPIO-loaded microbubbles for mediating and monitoring blood brain barrier permeation, Adv. Funct. Mater. 25 (1) (2015) 36 43. [54] B. Obermeier, R. Daneman, R. Ransohoff, Development, maintenance and disruption of the blood brain barrier, Nat. Med. 19 (12) (2013) 1584 1596. [55] S.J. Hollister, Porous scaffold design for tissue engineering, Nat. Mater. 5 (7) (2005) 518 524. [56] R. McKeon, et al., Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes, J. Neurosci. 11 (11) (1991) 3398 3411. [57] A. Misra, et al., Drug delivery to the central nervous system: a review, J. Pharm. Pharm. Sci. 6 (2003) 252 273. [58] G. Orive, et al., Biomaterials for promoting brain protection, repair and regeneration, Nat. Rev. Neurosci. 10 (9) (2009) 682 692. [59] X. Wang, et al., Hyaluronic acid-based scaffold for central neural tissue engineering, Interface Focus 2 (3) (2012) 278 291. [60] R.G. Ellis-Behnke, et al., Using nanotechnology to design potential therapies for CNS regeneration, Curr. Pharm. Des. 13 (24) (2007) 2519 2528. [61] D.B. Hoban, et al., The reduction in immunogenicity of neurotrophin overexpressing stem cells after intra-striatal transplantation by encapsulation in an in situ gelling collagen hydrogel, Biomaterials 34 (37) (2013) 9420 9429.

318

THERANOSTIC BIONANOMATERIALS

[62] X. Zhao, et al., Molecular self-assembly and applications of designer peptide amphiphiles, Chem. Soc. Rev. 39 (9) (2010) 3480 3498. [63] R.C. Assunção-Silva, et al., Hydrogels and cell based therapies in spinal cord injury regeneration, Stem. Cells Int. 2015 (2015) 24. [64] L.R. Nih, S.T. Carmichael, T. Segura, Hydrogels for brain repair after stroke: an emerging treatment option, Curr. Opin. Biotechnol. 40 (2016) 155 163. [65] C.C. Tate, et al., Laminin and fibronectin scaffolds enhance neural stem cell transplantation into the injured brain, J. Tissue Eng. Regen. Med. 3 (3) (2009) 208 217. [66] V.W. Nga, et al., Effects of polycaprolactone-based scaffolds on the blood brain barrier and cerebral inflammation, Tissue Eng. Part A 21 (3-4) (2015) 647 653. [67] W.M. Tian, et al., Hyaluronic acid-poly-D-lysine-based three-dimensional hydrogel for traumatic brain injury, Tissue Eng. 11 (3-4) (2005) 513 525. [68] S. Hou, et al., The repair of brain lesion by implantation of hyaluronic acid hydrogels modified with laminin, J. Neurosci. Methods 148 (1) (2005) 60 70. [69] F.Z. Cui, et al., Hyaluronic acid hydrogel immobilized with RGD peptides for brain tissue engineering, J. Mater. Sci. Mater. Med. 17 (12) (2006) 1393 1401. [70] M.C. Tate, et al., Biocompatibility of methylcellulose-based constructs designed for intracerebral gelation following experimental traumatic brain injury, Biomaterials 22 (10) (2001) 1113 1123. [71] T.Y. Cheng, et al., Neural stem cells encapsulated in a functionalized self-assembling peptide hydrogel for brain tissue engineering, Biomaterials 34 (8) (2013) 2005 2016. [72] J. Lam, et al., Delivery of iPS-NPCs to the stroke cavity within a hyaluronic acid matrix promotes the differentiation of transplanted cells, Adv. Funct. Mater. 24 (44) (2014) 7053 7062. [73] J. Zhong, et al., Hydrogel matrix to support stem cell survival after brain transplantation in stroke, Neuroreahabil. Neural Repair 24 (7) (2010) 636 644. [74] T.Y. Wang, et al., Promoting engraftment of transplanted neural stem cells/progenitors using biofunctionalised electrospun scaffolds, Biomaterials 33 (36) (2012) 9188 9197. [75] K.K. Bokara, et al., Biocompatability of carbon nanotubes with stem cells to treat CNS injuries, Anat. Cell Biol. 46 (2) (2013) 85 92. [76] E. Jan, N. Kotov, Successful differentiation of mouse neural stem cells on layer-by-layer assembled single-walled carbon nanotube composite, Nano Lett. 7 (5) (2007) 1123 1128. [77] J.L. McKenzie, et al., Decreased functions of astrocytes on carbon nanofiber materials, Biomaterials 25 (7-8) (2004) 1309 1317. [78] P. Galvan-Garcia, et al., Robust cell migration and neuronal growth on pristine carbon nanotube sheets and yarns, J. Biomater. Sci. Polym. Ed. 18 (10) (2007) 1245 1261. [79] S. Nune, et al., Nanoparticles for biomedical imaging, Expert Opin. Drug. Deliv. 6 (11) (2009) 1175 1194. [80] A.-M. Chacko, et al., Targeted delivery of antibody-based therapeutic and imaging agents to CNS tumors: crossing the blood brain-barrier divide, Expert Opin. Drug Deliv. 10 (7) (2013) 907 926. [81] J. Frigell, et al., 68Ga-labeled gold glyconanoparticles for exploring blood 2 brain barrier permeability: preparation, biodistribution studies, and improved brain uptake via neuropeptide conjugation, J. Am. Chem. Soc. 136 (1) (2014) 449 457. [82] A. Sawyer, T. Kyriakides, Nanoparticle-based evaluation of blood brain barrier leakage during the foreign body response, J. Neural Eng. 10 (1) (2013) 016013. [83] A. Smith, S. Nie, Next-generation quantum dots, Nat. Biotechnol. 27 (8) (2009) 732 733.

Chapter 13 • Theranostic Biomaterials for Regulation of the Blood Brain Barrier 319

[84] Y. Zhu, et al., Quantum dot-based nanoprobes for in vivo targeted imaging, Curr. Mol. Med. 13 (10) (2013) 1549 1567. [85] A. Bonoiu, et al., MMP-9 gene silencing by a quantum dot-siRNA nanoplex delivery to maintain the integrity of the blood brain barrier, Brain Res. 1282 (2009) 142 155. [86] Wahajuddin, S. Arora, Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers, Int. J. Nanomed. 7 (2012) 3445 3471. [87] R. Qiao, et al., Receptor-mediated delivery of magnetic nanoparticles across the blood brain barrier, ACS Nano 6 (4) (2012) 3304 3310. [88] M. Mahmoudi, et al., Superparamagnetic iron oxide nanoparticles: promises for diagnosis and treatment of multiple sclerosis, ACS Chem. Neurosci. 2 (3) (2011) 118 140. [89] D. Liu, et al., Effective PEGylation of iron oxide nanoparticles for high performance in vivo cancer imaging, Adv. Funct. Mater. 21 (8) (2011) 1498 1504. [90] J. Bulte, et al., Dextran-magnetite particles: contrast-enhanced MRI of blood brain barrier disruption in a rat model, Magn. Reson. Med. 23 (2) (1992) 215 223. [91] D. Liu, et al., Magnetic resonance imaging of post-ischemic blood brain barrier damage with PEGylated iron oxide nanoparticles, Nanoscale 6 (24) (2014) 15161 15167. [92] X. Feng, et al., Central nervous system toxicity of metallic nanoparticles, Int. J. Nanomed. 10 (2015) 4321 4340.

14 Upconversion Nanomaterials for Near-infrared Light-Mediated Theranostics Lili Xie1, Caihou Lin2, Qiushui Chen3, Huang-Hao Yang1 1

COLLEGE OF CH EMISTRY, FUZHOU UNIVERSITY, FUZHOU, P.R. CHINA 2 DEPART ME NT OF NEUROSURGERY , FUJIAN M EDICAL UNIVERSITY UNION HOSPITAL, FUZHOU, P.R. CHINA 3 DEPARTM ENT O F CHEM IST RY , NAT IONAL UNIVERSITY OF SINGAPORE, SINGAPORE, SINGAPORE

14.1 Introduction Nanotechnology-based theranostics have been rapidly developed for precise medicine through the use of luminescence nanoparticles as biolabeling probes for light-driven sensing, imaging, and therapy [1 4]. The luminescence nanoparticles enable the detection, visualization, and quantification of molecules or cells to enable more accurate disease diagnosis [5,6]. In addition, these luminescence nanoparticles provide an opportunity to achieve controllable noninvasive optical imaging for disease diagnosis and therapy [7]. For instance, the nanoparticles can be used as nanocarriers for controllable release of drugs upon light activation, and provide a powerful tool for light-mediated cancer therapy [8,9]. However, conventional downconversion luminescence nanomaterials, such as quantum dots, have limitations in biomedical applications as the associated Ultraviolet (UV) visible excitations exhibit autoluminescence background and shallow light penetration in tissues. In recent years, lanthanide-doped upconversion nanomaterials have increasingly emerged as a new class of nanoprobes that are able to convert two or more low-energy invisible near-infrared (NIR) photons into one high-energy visible photon [10]. Compared to conventional organic dyes and quantum dots, NIR-excitable upconversion nanoparticles (UCNPs) exhibit deep light penetration, excellent photostability, low toxicity, and avoided autofluorescence in biological tissues [11]. In this regard, upconversion nanoparticles have raised several interesting applications, such as biosensing, biological imaging, drug carriers, and photodynamic therapy (PDT) [12,13]. For example, Peng et al. reported a design of dyeconjugated photon upconverting nanoprobes for real-time monitoring hepatotoxicity in vivo [14]. Due to excellent light penetration and low autofluorescence of NIR excitation in deep tissues, the as-designed upconversion nanosensor can be used to achieve biomarker

Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00014-6 © 2019 Elsevier Inc. All rights reserved.

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detection for disease diagnosis. Meanwhile, the lanthanide-doped upconversion nanoparticles hold great promising for noninvasive light-mediated cancer therapy. One example by Zhang et al. [15] is to develop NIR-driven PDT. Mesoporous-silica-coated upconversion nanoparticles were used as an NIR nanotransducer and a carrier of photosensitizers (PS). The NIR-excitable nanoparticles allow for simultaneous activation of two PS to enhance PDT. Moreover, the nanoparticles have led to a breakthrough in the field of deep-tissue brain simulation through upconversion-mediated optogenetic therapy [16]. Upconversion nanoparticles can be specially designed to convert NIR light from outside the brain into the local emission visible light, enabling noninvasive optogenetic therapy. Thus, the emerging upconversion nanoparticles are very attractive for use as theranostic bionanomaterials. In this chapter, we introduce the fundamentals of lanthanide-doped upconversion nanoparticles and their utility in NIR light-mediated theranostics. We first talk about the basic properties of these nanoparticles, such as luminescence mechanism, optical properties, and materials engineering. This will help in understanding the importance and merits of the upconversion nanoparticles used in biomedical studies. Next, we present an overview of the popular applications of upconversion nanoparticles in diagnosis and therapy: biomarker sensing, optical imaging, and medical therapy. These sections will also introduce some representative examples to discuss the unique advantages of using upconversion nanoparticles as biological nanoprobes and optical nanotransducers for theranostic applications. Furthermore, we will discuss biosafety considerations of lanthanide-doped nanoparticles and their potential risks in biomedical applications. Finally, we give a summary of the chapter and a perspective in these research fields.

14.2 Upconversion Nanoparticle Design Considerations 14.2.1 Luminescence Mechanism of Upconversion Nanoparticles Photon upconversion is a nonlinear optical phenomenon in which two or more low-energy (long-wavelength) photons are converted into one high-energy (short-wavelength) light emission [17,18]. The optical properties and luminescence mechanism of these nanomaterials are shown in Fig. 14 1 [11]. In particular, inorganic upconversion nanocrystals have been intensely studied as a type of important material for photon upconversion. Lanthanidedoped upconversion nanocrystals are commonly composed of a crystalline host matrix doped with lanthanide ions. Upconversion nanocrystals typically require doping trivalent Yb31 or Nd31 ions as sensitizers to absorb NIR photons and transfer energy to activators, such as Tm31, Er31, and Ho31 ions [19]. For an efficient upconversion to proceed, the upconversion mechanism mainly involves a long-lived intermediate state to store excitation energies, which usually exists in energy transfer upconversion and energy migration upconversion [20]. In a typical process, energy transfer can occur between sensitizers and activators to dominate high-efficiency upconversion luminescence, a process where the sensitizers harvest the NIR light and then transfer the absorbed energy to the activator luminescence centers for visible emission. Conventional high-efficiency lanthanide-doped upconversion

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FIGURE 14–1 Lanthanide-doped nanoparticles and photon upconversion. (A) Three distinct classes of luminescent probes including organic dyes, quantum dots, and lanthanide-doped nanoparticles. (B) Mechanism of photon upconversion in lanthanide-doped upconversion nanocrystals. © 2015 Springer Nature.

nanoparticles are based on a co-doping of Yb31 or Nd31 ions as a sensitizer, which are excitable under a single wavelength, either 980 or 808 nm, laser source [21,22]. Notably, doping high concentrations of sensitizers or activators is known to exhibit severe luminescence quenching caused by cross-relaxation or energy migration to lattices and surface defects [23,24]. These inherent limitations in lanthanide-doped nanocrystals have restricted the use of photon upconversion at a low doping level of lanthanide ions and single-band laser excitation. Recently, heavily doped upconversion nanoparticles have been developed through material engineering to overcome the effects of concentration quenching [25]. For example, a new class of lanthanide-doped nanoparticles with bright upconversion luminescence was developed through Er31-based self-sensitization upconversion [26]. Erbiumenriched core shell nanoparticles exhibit very strong energy migration between Er31 ions, and unlock concentration quenching upon surface coating. These nanocrystals offer unique

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features of multiexcitations, bright red and NIR luminescence for sensitive biological imaging. As discussed earlier, fundamental investigations into the upconversion luminescence mechanism can offer a pathway to design and develop a series of desired nanocrystals for various applications.

14.2.2 Synthesis and Surface Engineering To utilize upconversion nanoparticles as bioprobes or nanotransducers, it is essential to precisely control the size, shape, toxicity, optical properties, and luminescence efficiency of the nanoparticles [13,27]. In this regard, many techniques have been developed to synthesize upconversion nanocrystals with specific properties. The hydrothermal method is a general solution-based approach to facilitate preparation of lanthanide-doped upconversion nanocrystals [28,29]. Through doping appropriate lanthanide ions, it is easy to control the size, morphology, and optical properties of upconversion nanoparticles with advantages of low cost and mass production. However, the use of thermal synthesis is difficult to fabricate small upconversion nanoparticles with high-efficiency luminescence capability and good dispersibility. Alternatively, solvothermal methods of coprecipitation and decomposition are developed to synthesize ultrasmall, high-crystallinity, and bright upconversion nanocrystals [30 33]. Using this method, it is also convenient to control the size, morphology, and emission of the lanthanide-doped nanocrystals. Especially, fluoride-based lanthanide nanocrystals such as NaYF4, NaGdF4, and NaLuF4 are a class of high-quality host matrix with better performance of desired nanostructures. It is worth mentioning that the design of core shell architecture has been demonstrated to be very effective in enhancing upconversion luminescence by preventing a surface-quenching effect or energy migration to the surface of the nanoparticles [34]. In addition, the design of core shell nanoparticles also allows for better management of photon energy transfer to overcome cross-relaxation-induced luminescence quenching [35]. In addition to the methods of nanoparticle synthesis, surface engineering is vital in designing biocompatible and functional upconversion nanoparticles. For example, a simple procedure can be developed to prepare amine-functionalized UCNPs through hydrothermal microemulsion assisted with 6-aminohexanoic acid (Fig. 14 2) [36]. This leads to excellent dispersibility in water and allows conjugation with targeted molecules for biological applications. Silica-coated upconversion nanoparticles have been developed as a versatile platform for the development of efficient theranostics [37,38]. The coating of silica can make the upconversion nanocrystals have low toxicity and be easy to modify with biological molecules, which is important for biomedical applications. Alternatively, ligand engineering is also a common approach to fabricate a functional group on the surface of upconversion nanoparticles. A typical method is to use the ligand exchange reaction to replace hydrophobic ligands with a wide variety of hydrophilic molecules on the surface of upconversion nanoparticles, such as poly-(ethylene glycol) (PEG) diacid [39], citrate [40], and polyacrylic acid [41,42]. Additionally, surface functionalization of upconversion nanocrystals can be also achieved by electrostatic later-by-layer assembly, ligand attraction, and surface conjugation [43 45].

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FIGURE 14–2 Synthesis of biocompatible upconversion nanoparticles (UCNPs) via a modified hydrothermal microemulsion route. © 2009 Elsevier Ltd.

Indeed, the strategies of surface engineering make the upconversion nanoparticles more affordable for various applications in theranostics.

14.2.3 Upconversion Luminescence Tuning Fine tuning of upconversion luminescence is highly desirable to meet the requirement of their biological applications, such as multiplexed molecular labeling, light-triggered drug release, optical imaging, and PDT. A typical strategy to tune the upconversion emission involves adjustment of dopant concentrations of lanthanide activators or nanocrystal host. Under a single NIR excitation at 980 or 808 nm, the upconversion emission can be well tailored in the field of UV, visible, and NIR. For example, upconversion nanoparticles can be designed to emit a number of colors through doping with different activators (Tm31, Er31, Ho31, Eu31, or Tb31 ions) available for luminescence centers. Alternatively, tuning of upconversion emission can be realized through controlling dopant concentration and composition, excitation power density, and host lattice of nanocrystals [11]. For example, Ho31/Ce31codoped nanoparticles were found to exhibit tunable emission color from green to red through a pulsed laser excitation (Fig. 14 3) [46]. In addition, core shell nanocrystals can be designed to achieve multicolor emission under two different excitations at 980 and 808 nm [47]. On a separate note, Mn21-doped upconversion nanoparticles are found to exhibit time-resolved luminescence emission [48]. These studies provide a convenient strategy to manipulate upconversion luminescence emission for various biomedical applications.

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FIGURE 14–3 Multicolor tuning in upconversion nanocrystals. (A) Design of core shell nanocrystals for tunable emission color upon NIR irradiation. (B) Emission spectra of the upconversion nanocrystals using different pulse durations. (C) Photographs of multicolor luminescence tuning under 980- and 808-nm excitations. (D) Corresponding color gamut of the emission colors. NIR, Near-infrared. © 2015 Springer Nature.

14.3 Upconversion Nanoprobes for Biosensing 14.3.1 In Vitro Assays of Biomarkers NIR-excitable upconversion nanoparticles have emerged as a kind of ideal luminescence nanoprobe for biosensing due to having tissue-penetrating NIR excitation light, nonblinking luminescence, high photostability, and excellent biocompatibility. As compared to conventional fluorophores, the use of lanthanide-doped nanoparticles for labeling thus offers many advantages for in vitro assays of biomarkers, such as high signal-to-noise ratios and improved limits of detection. By taking advantage of the unique NIR light-converting properties, the multiplexing emissions at UV, visible, and NIR ranges can be utilized to selectively image the specific disease localization and to remotely activate the biomolecule activities in a highly spatiotemporal resolution. The upconversion nanoprobe-based bioassays can be divided into two different methods: heterogeneous and homogeneous assays. In particular, the heterogeneous assay is a method to detect target analytes through recognition molecules

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FIGURE 14–4 Homogeneous sandwich hybridization assay. The UCP nanoparticles were conjugated with the capture (C) oligonucleotides that are specific to the single-stranded target (T) sequence of HLA-B27 or β-actin. The formation of sandwich complex can induce the upconversion LRET signal. AF546, Alexa Fluor 546; AF700, Alexa Fluor 700; LRET, lanthanide resonance energy transfer; UCP, upconverting phosphor. © 2009 The Royal Society of Chemistry.

functionalized on a solid substrate. For example, upconversion nanoparticles can be used as labeling agents for immunoassays [49,50]. Y2O2S:Yb31/Er31 nanoparticles have been developed as luminescence nanoprobes to achieve highly sensitive detection of human chorionic gonadotropin [49]. Unlike heterogeneous assays, homogeneous assays do not need to separate unbound labels through the use of binding-modulated signals via luminescence resonance energy transfer (LRET) between a donor and an acceptor. This method is able to achieve multiplexed sensing through multipeak emission profiles of upconversion nanoparticles. For instance, Rantanen et al. developed a dual-parameter sandwich-hybridization assay to detect two different target-oligonucleotide sequences (Fig. 14 4) [51]. However, due to the limitation of the low efficiency of the LRET process, the detection sensitivity of upconversion-based homogenous assay is still limited.

14.3.2 In Vivo Detection of Biomolecules Since upconversion nanoparticles can be illuminated using deep tissue-penetrable NIR light, this offers an opportunity to realize in vivo detection of biomolecules in animals. For example, an upconversion nanoplatform can be designed to selectively localize tumor sites for early cancer diagnosis [14]. Upon using upconversion nanoparticle doping with different elements, NIR light-mediated luminescence nanoparticles can be used for multifunctional sensing. Xing et al. developed a tumor microenvironment-sensitive upconversion platform to perform localization of particles at the tumor site through tumor-specific cathepsin protease reactions (Fig. 14 5) [52]. Under 808-nm light excitation, upconversion luminescence was

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FIGURE 14–5 In vivo detection of the tumor microenvironment using peptide-premodified UCNs. The UCNs were triggered to accumulate into the tumor sites by tumor-specific CtsB enzyme reactions due to cleavage of the peptides on the particle surface. This leads to enhanced upconversion emission under 808-nm NIR irradiation. CtsB, Cathepsin B; NIR, near-infrared; UCN, upconversion nanocrystals. © 2016 Springer Nature.

enhanced by accumulated upconversion nanoparticles, thus allowing for in vivo detection of tumor biomarkers in tumor sites. Meanwhile, upconversion sensors can be developed to determine hepatotoxicity in vivo by chromophore-conjugated upconversion nanoparticles based on NIR-mediated luminescence signal [14]. Upconversion nanoprobes are designed to accumulate in the liver. The presence of ONOO , a species of molecule indicating hepatotoxicity, can lead to activating energy transfer from nanoprobes to the chromophore. On the basis of this principle, the upconversion nanoparticles were utilized to achieve real-time in vivo hepatotoxicity monitoring. By taking advantage of excellent light penetration in tissues, the NIR-excitable nanoprobes can provide a convenient strategy to assess the hepatotoxicity of drugs. Thus, the upconversion nanomaterials enable the optical sensors to be more selective, more sensitive, and less expensive than existing methods.

14.4 In Vivo Bioimaging Using Upconversion Nanoparticles 14.4.1 Near-Infrared Light-based Optical Imaging Optical imaging provides great opportunities to spatiotemporally identify morphological functions in living organisms, and they have been well recognized as extremely promising imaging platforms for preclinical studies. A clear and real-time visualization of physiological events through these imaging modalities to provide unique insight into functions of living tissues associated with various diseases has been increasingly dependent on the availability of robust imaging probes. NIR biological nanoprobes have been rapidly developed for in vivo fluorescence-based optical imaging, due to their excellent light penetration and low autofluorescence to the biological samples. The traditional organic dyes as probes have been demonstrated to exhibit photobleaching for long-term optical imaging. Lanthanide-doped nanocrystals have been recognized as a promising NIR nanoprobe. Upconversion

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nanoparticles can be used for in vivo optical imaging with better deep tissue penetration than conventional quantum dots [53,54]. For deeper in vivo optical imaging, upconversion emission with red or NIR light (600 1100 nm) is preferred as it will reduce light scattering and absorbance in animal tissues. For example, upconversion nanoparticles with NIR-to-NIR upconversion have been reported to achieve optical in vivo imaging with a light penetration depth of up to 2 cm in animals [55]. The unique property of multicolor emission of upconversion nanoparticles renders us to achieve simultaneous optical imaging of different organisms. In vivo multicolor imaging was first demonstrated using NaYF4:Yb/Er/La nanorods [56]. It should be noted that the use of NIR laser excitations with a wavelength of 980 nm commonly exhibits severe heating effects on tissues. For this reason, it is of great interest to develop Nd31-sensitized upconversion nanoparticles, as excitation at 808 nm can be used to minimize the photothermal effects. Another strategy was also proposed to reduce the illumination time of NIR laser through a combination of persistence luminescence nanoparticles with NIR-triggered upconversion nanoparticles. Typically, persistent luminescence nanomaterials can store the excitation energies and then slowly release the luminescence emission. To reduce the heat influence through avoiding the use of continuous laser irradiation, NIR-excited upconversion nanoparticles are able to transfer their energies to the persistence luminescence nanoparticles, and emit NIR light at 700 nm for in vivo imaging [57]. Er31-sensitized upconversion nanocrystals (NaErF4@NaYF4) were further demonstrated to be excitable under 808-, 980-, or 1532-nm laser sources, thus they can serve as a kind of ideal NIR nanoprobe for in vivo imaging (Fig. 14 6) [26].

14.4.2 Upconversion Nanoparticles for Multimodel Bioimaging Upconversion nanoparticles have been also developed as contrast agents for multimodel bioimaging applications [58,59]. For example, it is easy to design multifunctional lanthanidedoped nanoparticles to combine optical imaging with other imaging modalities, such as computed tomography (CT), positron-emission tomography (PET), and magnetic resonance imaging (MRI). The multifunctional nanoparticles can be used as multimodal agents to help visualize precise medical diagnosis. Gd31-based upconversion nanoparticles exhibit a combination of magnetic and optical properties in a single nanoparticle [60]. These nanoparticles are affordable to perform in vivo optical imaging and MRI, where MRI is able to offer an excellent spatial resolution and deep tissue imaging. For example, Zeng et al. demonstrated that NaLuF4:Yb31, Er31, Gd31 nanocrystals can be used as bifunctional contrast agents for in vivo optical imaging and MRI [61]. Meanwhile, Fe3O4 nanoparticles have also been employed as good candidates for MRI, which can be coupled with upconversion nanoparticles for optical imaging and MRI for dual-model applications [62]. Since the lanthanide-doped upconversion nanoparticles have efficient X-ray absorption, they have been used as contrast agents to achieve CT for efficient noninvasive medical diagnosis, by combination with upconversion-based optical imaging [63]. In addition, when doped with radionuclide in nanoparticles such as 18F or 153Sm, the lanthanide-based materials are able to achieve PET and optical imaging simultaneously [64,65]. Many studies have

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FIGURE 14–6 Er31-sensitized upconversion nanocrystals for multiwavelength-excitable optical imaging. (A) Emission spectra of Er31-based nanoparticles upon 808-, 980-, and 1532-nm excitations. (B) In vivo optical imaging using silica-coated NaErF4:Tm@NaYF4 nanoparticles with excitations at 808, 980, and 1532 nm. (C) Optical windows related with luminescence profiles for as-synthesized nanocrystals. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

demonstrated the availability of lanthanide-doped nanocrystals as nanoprobes for multimodel bioimaging, including MRI, CT, and optical imaging (Fig. 14 7) [66]. The development of upconversion nanoparticles with multimodel bioimaging holds great promise for image-guided therapy in precise medicine.

14.5 Photon Upconversion-Mediated Medical Therapy 14.5.1 Near-Infrared Light-Triggered Drug Delivery Over recent years, upconversion nanoparticles have been developed as nanocarriers for drug delivery in medical therapies. UV visible light emission under NIR excitations is able to achieve controllable release of drugs on demand [67 69]. Upconversion-based nanocarriers can be designed through physical absorption and porous loading of drugs, for example, physical absorption by its porous nanostructure and hydrophobic interaction with hydrophobic drugs. Meanwhile, upconversion nanoparticles with a shell layer of mesoporous structure have been widely designed to load drugs [70,71]. Upon NIR irradiation, the loaded drugs can

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FIGURE 14–7 Dye-sensitized upconversion nanoparticles coated with a mesoporous silica layer and loaded with PS of Ce6 and MC540 for imaging-guided PDT. PDT, Photodynamic therapy; PS, photosensitizers. © 2017 American Chemical Society.

be control-released to kill target cells for therapy purposes. One drawback to this technique is that the size of the nanoparticles is usually too large (.10 nm) and thus the nanoparticles cannot be easily excreted from the animal body. Another strategy is to load drugs with upconversion nanoparticles through hydrophobic interaction. This method can lead to reduced size nanocarriers when injected into the animal body. To achieve this goal, upconversion nanoparticles are modified with amphiphilic molecules and form a hydrophobic layer on their surface. Meanwhile, to realize targeted release of drugs in tumors, a pH-responsive upconversion nanoparticle system has been explored, since the properties of molecules can be designed to be response with pH environments. For example, a chemotherapeutic drug doxorubicin (DOX) was physically absorbed on PEGlyated upconversion nanoparticles (Fig. 14 8) [72]. The DOX drugs can become more water-soluble at low pH value at the tumor site, and targeted drug delivery can be implemented. This technique is of practical significance for clinical cancer therapy.

14.5.2 Near-Infrared Light-Activated Photodynamic Therapy Therapeutic application of upconversion nanoparticles on NIR-triggered PDT is a technique that uses visible light to activate surrounding PS molecules, which are capable of generating singlet oxygen (1O2) to kill cells [73]. The conventional PDT technique is usually limited by the short penetration depth because of the use of visible light for photoactivation. NIRmediated PDT via upconversion nanoparticles has been intensely studied in recent years, as the use of NIR light excitation allows for much better light penetration in tissues for therapy purposes. Zhang et al. reported the study of upconversion-mediated in vitro PDT through

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FIGURE 14–8 Schematic of upconversion nanoparticle-mediated drug delivery. (A) Oleic acid-capped upconversion nanoparticle. (B) PEGlyated upconversion nanoparticles. (C) DOX drugs physically loaded on the nanoparticle surface via hydrophobic interaction. (D) pH-triggered controllable release of DOX from nanoparticles. DOX, Doxorubicin; PEG, poly-(ethylene glycol). © 2010 Elsevier Ltd.

the use of silica-coated upconversion nanoparticles coupled with PS [74]. Upon NIR light irradiation, PS-loaded NaYF4Yb/Er@SiO2 nanoparticles can produce singlet oxygen to kill cells on demand. The NIR-activated PDT exhibits several merits during therapy, including deep tissue penetration, cost effectiveness, and relatively specific tumor treatment. After that, upconversion-based in vivo PDT was demonstrated in mice. PS (Ce6) were absorbed onto PEGylated nanoparticles and injected into BABL/C mice with 4T1 murine breast cancer tumors [75]. Upon NIR irradiation, the size of tumors was successfully reduced by up to 70%. To improve the PDT efficiency, multicolor-emitting upconversion nanoparticles were employed to achieve activation of two PS under a single NIR excitation (Fig. 14 9) [15]. This technique was found to be able to efficiently inhibit tumor growth, and serve as a promising approach for noninvasive cancer therapy. In addition, the capability of PS loading is also important for efficient PDT. To achieve this, mesoporous silica-coated upconversion nanoparticles were prepared to increase the amount of PS [37]. This design was found to simultaneously enhance the loading capability and photon energy transfer efficiency, and thus significantly improve the PDT efficiency. On a separate note, the design and synthesis of high-quality upconversion nanoparticles is also critical to ensure PDT efficiency. This can be promoted by a fundamental investigation of the materials.

14.5.3 Near-Infrared Light-Mediated Optogenetic Therapy Optogenetics has become an important technique which uses light to control cells in living tissue for treatment of neurological disorders [76,77]. A specific light can be applied to

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FIGURE 14–9 (A, B) Transmission electron microscopy images of the mesoporous-silica-coated NaYF4:Yb/Er nanoparticles at low (A; scale bar, 100 nm) and high magnification (B; scale bar, 50 nm). (C) Fluorescence spectrum of the nanoparticles upon 980-nm excitation and absorption spectra of PS. (D) Upconversion-medicated PDT through remote-controlled nanotransducers. PDT, Photodynamic therapy; PS, photosensitizers. © 2012 Springer Nature.

spatiotemporally precise control of light-sensitive molecular receptors (Fig. 14 10) [78]. Most of the photoreceptors in cells are only able to be activated by visible light. However, the inability of visible light to penetrate into deep tissues in the brain is a big challenge for practical applications of this technique. Currently, deep brain stimulation commonly needs an electrode to be inserted directly into the brain. NIR-excitable upconversion nanoparticles have been demonstrated to hold promising in deep tissue penetration. In 2011, the concept of upconversion-mediated optogenetics was first proposed by Deisseroth et al. [79]. An NIRmediated optogenetic technique was further developed for remote control of Ca21 oscillations and Ca21-responsive gene expression, which can regulate the function of cells. In particular, upconversion nanoparticles were employed to convert NIR light into visible emissions for optogenetic operation [80]. In 2017, Chen et al. reported that upconversion nanoparticles can be used as optogenetic actuators of transcranial NIR light to stimulate neurons in deep brain [16]. NIR light-mediated optogenetics is able to evoke release of dopamine from neurons, and induce brain oscillations [81]. This study demonstrated that upconversion-mediated optogenetics can be a promising noninvasive nanotechnologyassisted strategy for optical control of neuronal activity, and might eventually offer an

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FIGURE 14–10 Light-mediated optogenetics to modulate membrane voltage potential. © 2011 Springer Nature.

opportunity for clinical applications to treat neuronal diseases, such as Parkinson's disease and paralysis. When considering practical applications, the risks of the NIR laser could be a major problem for clinical applications. The heating effects of the 980-nm laser can also become a notable problem for practical therapy applications as it can cause possible tissue damage if not well controlled.

14.6 Toxicity Studies of Upconversion Nanoparticles Since the lanthanide-doped nanomaterials have been widely studied for theranostic applications, it places a high demand on the evaluation of their biological toxicity on cells and animals. It should be noted that the leakage of lanthanide ions from the nanoparticles is likely to interact with cells and cause biological hazards. In a typical procedure, a general standardized cytotoxicity test is utilized to determine the influence of the upconversion nanoparticles on the cells, including cellular morphology and mitochondrial function (MTT and MTS assays). Most studies indicate that the lanthanide-doped nanoparticles have no obvious toxicity when incubated with a broad range of cell lines. For example, it is reported that carboxyl- and amino-functionalized upconversion nanoparticles do not exhibit toxicity after incubation with human osteosarcoma cells [82]. However, Tian et al. found that ligand-free

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FIGURE 14–11 Illustration of lanthanide-doped nanoparticle-induced cell apoptosis and autophagy through intracellular ATP deprivation. ATP, Adenosine triphosphate. © 2015 American Chemical Society.

lanthanide-doped nanoparticles can induce intracellular adenosine triphosphate (ATP) deprivation and cause a significant decrease in cell viability after incubation for 7 days [83]. The long-term cytotoxicity of lanthanide-doped nanoparticles was systematically evaluated by monitoring cell viability, ATP level, and cell membrane integrity, respectively. Particleinduced cell death was likely to be primarily mediated via the interaction between the phosphate group of cellular ATP and the nanoparticle (Fig. 14 11). In vivo evaluation of the toxicity of upconversion nanoparticles can be performed by animal studies since the uptake of nanoparticles in animal bodies is much more complex than in cells. The biological distribution of upconversion nanoparticles in different animal organs has been examined after injection at different time intervals. It was found that most of the upconversion nanoparticles can be excreted by rats after 7 days. Zhang et al. showed no weight loss or abnormal behavior when silica-coated NaYF4:Yb/Er nanoparticles were injected intravenously into healthy rats with a dosage of 10 mg/kg body weight after 7 days [84]. More recently, Lin et al. observed that the ligand-free lanthanide-doped nanoparticles can cause a negative influence on neurobehavioral performance and morphological signs of brain damage in mice [85,86]. Through histopathological analysis and tests of spatial recognition memory, the experimental evidence suggested a potential risk of toxicity and damage using bare rare-earth nanoparticles on mouse brain. Long-term toxicity studies concerning particle size and surface chemistry are necessary to be further carried out to assess the suitability of the upconversion nanoparticles for in vivo biological applications.

14.7 Conclusion and Outlook In this chapter, we have introduced the development of upconversion nanomaterials and their applications in NIR light-mediated theranostic applications. Due to the outstanding

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properties of lanthanide-doped upconversion nanoparticles, they can afford various biomedical applications with unique advantages including multicolor emission capability under single-wavelength excitation, high signal-to-noise ratio, exceptional biocompatibility, excellent chemical- and photostability and the ability to allow deep-tissue optical imaging. Since the pioneering work in the past 10 years, upconversion nanocrystals have held great promise for use in theranostic applications spanning biosensing to optical imaging and optical therapy. Notably, the development of NIR light-excitable nanoparticles has created many significant breakthroughs for biomedical therapy, including deep-tissue PDT and optogenetic therapy. Despite promising aspects, there still remain constraints associated with the practical applications of upconversion nanoparticles in theranostics. The penetration depth of NIR light in tissues is still very limited (about 1 cm), and the heating effects of using NIR laser are a potential risk for therapy applications. In this regard, the development of high-quality upconversion nanoparticles with high-efficiency luminescence yield and NIR-to-NIR conversion might be a future research direction. A new type of X-ray-excitable nanocrystal could also be promising to break the limit of light penetration in deep tissues for theranostic applications [87,88]. In addition, although lanthanide-doped upconversion nanoparticles have been demonstrated to have low toxicity in cells and animal bodies, much more effort is still required to be done to ensure the biological safety of long-term use of these materials.

References [1] J. Ahmad, et al., Nanotechnology based theranostic approaches in Alzheimer's disease management: current status and future perspective, Curr. Alzheimer Res. 14 (2017) 1164 1181. [2] N. Ahmed, H. Fessi, A. Elaissari, Theranostic applications of nanoparticles in cancer, Drug Discov. Today 17 (2012) 928 934. [3] Y.I. Park, et al., Theranostic probe based on lanthanide-doped nanoparticles for simultaneous in vivo dual-modal imaging and photodynamic therapy, Adv. Mater. 24 (2012) 5755 5761. [4] M.P. Melancon, M. Zhou, C. Li, Cancer theranostics with near-infrared light-activatable multimodal nanoparticles, Acc. Chem. Res. 44 (2011) 947 956. [5] T. Anani, P. Panizzi, A.E. David, Nanoparticle-based probes to enable noninvasive imaging of proteolytic activity for cancer diagnosis, Nanomedicine (Lond) 11 (2016) 2007 2022. [6] M. Dekaliuk, K. Pyrshev, A. Demchenko, Visualization and detection of live and apoptotic cells with fluorescent carbon nanoparticles, J. Nanobiotechnol. 13 (2015) 86. [7] S. Kunjachan, et al., Noninvasive optical imaging of nanomedicine biodistribution, ACS Nano 7 (2013) 252 262. [8] J. Shen, L. Zhao, G. Han, Lanthanide-doped upconverting luminescent nanoparticle platforms for optical imaging-guided drug delivery and therapy, Adv. Drug Deliv. Rev. 65 (2013) 744 755. [9] L.J. Chen, C.X. Yang, X.P. Yan, Liposome-coated persistent luminescence nanoparticles as luminescence trackable drug carrier for chemotherapy, Anal. Chem. 89 (2017) 6936 6939. [10] F. Wang, X. Liu, Recent advances in the chemistry of lanthanide-doped upconversion nanocrystals, Chem. Soc. Rev. 38 (2009) 976 989.

Chapter 14 • Upconversion Nanomaterials

337

[11] B. Zhou, B. Shi, D. Jin, X. Liu, Controlling upconversion nanocrystals for emerging applications, Nat. Nanotechnol. 10 (2015) 924 936. [12] F. Wang, D. Banerjee, Y. Liu, X. Chen, X. Liu, Upconversion nanoparticles in biological labeling, imaging, and therapy, Analyst 135 (2010) 1839 1854. [13] Z. Gu, et al., Recent advances in design and fabrication of upconversion nanoparticles and their safe theranostic applications, Adv. Mater 25 (2013) 3758 3779. [14] J. Peng, et al., Real-time in vivo hepatotoxicity monitoring through chromophore-conjugated photonupconverting nanoprobes, Angew. Chem. Int. Ed 56 (2017) 4165 4169. [15] N.M. Idris, et al., In vivo photodynamic therapy using upconversion nanoparticles as remote-controlled nanotransducers, Nat. Med. 18 (2012) 1580 1585. [16] S. Chen, et al., Near-infrared deep brain stimulation via upconversion nanoparticle-mediated optogenetics, Science 359 (2018) 679 684. [17] F. Auzel, Upconversion and anti-Stokes processes with f and d ions in solids, Chem. Rev. 104 (2004) 139 173. [18] M. Haase, H. Schafer, Upconverting nanoparticles, Angew. Chem. Int. Ed 50 (2011) 5808 5829. [19] X. Liu, C.H. Yan, J.A. Capobianco, Photon upconversion nanomaterials, Chem. Soc. Rev. 44 (2015) 1299 1301. [20] F. Wang, et al., Tuning upconversion through energy migration in core shell nanoparticles, Nat. Mater. 10 (2011) 968 973. [21] Y.F. Wang, et al., Nd31-sensitized upconversion nanophosphors: efficient in vivo bioimaging probes with minimized heating effect, ACS Nano 7 (2013) 7200 7206. [22] X. Xie, et al., Mechanistic investigation of photon upconversion in Nd31-sensitized core shell nanoparticles, J. Am. Chem. Soc. 135 (2013) 12608 12611. [23] Y. Liu, et al., Amplified stimulated emission in upconversion nanoparticles for super-resolution nanoscopy, Nature 543 (2017) 229 233. [24] D.J. Gargas, et al., Engineering bright sub-10-nm upconverting nanocrystals for single-molecule imaging, Nat. Nanotechnol. 9 (2014) 300 305. [25] S. Wen, et al., Advances in highly doped upconversion nanoparticles, Nat. Commun. 9 (2018) 2415. [26] Q. Chen, et al., Confining excitation energy in Er31-sensitized upconversion nanocrystals through Tm31mediated transient energy trapping, Angew. Chem. Int. Ed 56 (2017) 7605 7609. [27] F. Wang, et al., Simultaneous phase and size control of upconversion nanocrystals through lanthanide doping, Nature 463 (2010) 1061 1065. [28] W. Ren, et al., Lanthanide ion-doped GdPO4 nanorods with dual-modal bio-optical and magnetic resonance imaging properties, Nanoscale 4 (2012) 3754 3760. [29] N. Bogdan, et al., Bio-functionalization of ligand-free upconverting lanthanide doped nanoparticles for bio-imaging and cell targeting, Nanoscale 4 (2012) 3647 3650. [30] A. Dong, et al., A generalized ligand-exchange strategy enabling sequential surface functionalization of colloidal nanocrystals, J. Am. Chem. Soc. 133 (2011) 998 1006. [31] M. Saboktakin, et al., Metal-enhanced upconversion luminescence tunable through metal nanoparticlenanophosphor separation, ACS Nano 6 (2012) 8758 8766. [32] H.X. Mai, et al., High-quality sodium rare-earth fluoride nanocrystals: controlled synthesis and optical properties, J. Am. Chem. Soc. 128 (2006) 6426 6436. [33] J.C. Boyer, F. Vetrone, L.A. Cuccia, J.A. Capobianco, Synthesis of colloidal upconverting NaYF4 nanocrystals doped with Er31, Yb31 and Tm31, Yb31 via thermal decomposition of lanthanide trifluoroacetate precursors, J. Am. Chem. Soc. 128 (2006) 7444 7445.

338

THERANOSTIC BIONANOMATERIALS

[34] S. Han, R. Deng, X. Xie, X. Liu, Enhancing luminescence in lanthanide-doped upconversion nanoparticles, Angew. Chem. Int. Ed 53 (2014) 11702 11715. [35] Q. Su, et al., The effect of surface coating on energy migration-mediated upconversion, J. Am. Chem. Soc. 134 (2012) 20849 20857. [36] L.Q. Xiong, et al., Synthesis, characterization, and in vivo targeted imaging of amine-functionalized rareearth up-converting nanophosphors, Biomaterials 30 (2009) 5592 5600. [37] H.S. Qian, H.C. Guo, P.C. Ho, R. Mahendran, Y. Zhang, Mesoporous-silica-coated up-conversion fluorescent nanoparticles for photodynamic therapy, Small 5 (2009) 2285 2290. [38] J.N. Liu, W.B. Bu, J.L. Shi, Silica coated upconversion nanoparticles: a versatile platform for the development of efficient theranostics, Acc. Chem. Res. 48 (2015) 1797 1805. [39] G.M.C. Guang Shun Yi, Synthesis of hexagonal-phase NaYF4:Yb,Er and NaYF4:Yb,Tm nanocrystals with efficient up-conversion fluorescence, Adv. Funct. Mater. 16 (2006) 2324 2329. [40] T.Y. Tianye Cao, Yuan Gao, Yang Yang, He Hu, Fuyou Li, Water-soluble NaYF4:Yb/Er upconversion nanophosphors: synthesis, characteristics and application in bioimaging, Inorg. Chem. Commun. 13 (2010) 392 394. [41] M. Kamimura, D. Miyamoto, Y. Saito, K. Soga, Y. Nagasaki, Design of poly(ethylene glycol)/streptavidin coimmobilized upconversion nanophosphors and their application to fluorescence biolabeling, Langmuir 24 (2008) 8864 8870. [42] S.A. Hilderbrand, F. Shao, C. Salthouse, U. Mahmood, R. Weissleder, Upconverting luminescent nanomaterials: application to in vivo bioimaging, Chem. Commun. (2009) 4188 4190. [43] O. Ehlert, R. Thomann, M. Darbandi, T. Nann, A four-color colloidal multiplexing nanoparticle system, ACS Nano 2 (2008) 120 124. [44] M. Wang, et al., Immunolabeling and NIR-excited fluorescent imaging of HeLa cells by using NaYF4:Yb, Er upconversion nanoparticles, ACS Nano 3 (2009) 1580 1586. [45] N.J. Johnson, N.M. Sangeetha, J.C. Boyer, F.C. van Veggel, Facile ligand-exchange with polyvinylpyrrolidone and subsequent silica coating of hydrophobic upconverting beta-NaYF4:Yb31/Er31nanoparticles, Nanoscale 2 (2010) 771 777. [46] R. Deng, et al., Temporal full-colour tuning through non-steady-state upconversion, Nat. Nanotechnol. 10 (2015) 237 242. [47] H.Z. Hongli Wen, Xian Chen, Tak Fu Hung, Beilei Wang, Guangyu Zhu, Siu Fung Yu, et al., Upconverting near-infrared light through energy management in core shell shell nanoparticles, Angew. Chem. Int. Ed. 52 (2013) 13419 13423. [48] S. Han, et al., Multicolour synthesis in lanthanide-doped nanocrystals through cation exchange in water, Nat. Commun. 7 (2016) 13059. [49] J. Hampl, et al., Upconverting phosphor reporters in immunochromatographic assays, Anal. Biochem. 288 (2001) 176 187. [50] P.L. Corstjens, et al., Up-converting phosphor technology-based lateral flow assay for detection of Schistosoma circulating anodic antigen in serum, J. Clin. Microbiol. 46 (2008) 171 176. [51] T. Rantanen, et al., Upconverting phosphors in a dual-parameter LRET-based hybridization assay, Analyst 134 (2009) 1713 1716. [52] X. Ai, et al., In vivo covalent cross-linking of photon-converted rare-earth nanostructures for tumour localization and theranostics, Nat. Commun. 7 (2016) 10432. [53] G. Chen, H. Qiu, P.N. Prasad, X. Chen, Upconversion nanoparticles: design, nanochemistry, and applications in theranostics, Chem. Rev. 114 (2014) 5161 5214.

Chapter 14 • Upconversion Nanomaterials

339

[54] R. Wang, X. Li, L. Zhou, F. Zhang, Epitaxial seeded growth of rare-earth nanocrystals with efficient 800 nm near-infrared to 1525 nm short-wavelength infrared downconversion photoluminescence for in vivo bioimaging, Angew. Chem. Int. Ed. 53 (2014) 12086 12090. [55] A. Nadort, et al., Quantitative imaging of single upconversion nanoparticles in biological tissue, PLoS One 8 (2013) e63292. [56] M.L. Xuefeng Yu, M. Xie, L. Chen, Y. Li, Q. Wang, Dopant-controlled synthesis of water-soluble hexagonal NaYF4 nanorods with efficient upconversion fluorescence for multicolor bioimaging, Nano Res. 3 (2010) 51 60. [57] X. Qiu, et al., Hybrid nanoclusters for near-infrared to near-infrared upconverted persistent luminescence bioimaging, ACS Appl. Mater. Interfaces 9 (2017) 32583 32590. [58] H. Deng, S. Huang, C. Xu, Intensely red-emitting luminescent upconversion nanoparticles for deeptissue multimodal bioimaging, Talanta 184 (2018) 461 467. [59] X. Zhu, et al., Core-shell Fe3O4@NaLuF4:Yb,Er/Tm nanostructure for MRI, CT and upconversion luminescence tri-modality imaging, Biomaterials 33 (2012) 4618 4627. [60] C. Liu, et al., Magnetic/upconversion fluorescent NaGdF4:Yb,Er nanoparticle-based dual-modal molecular probes for imaging tiny tumors in vivo, ACS Nano 7 (2013) 7227 7240. [61] J.X. Songjun Zeng, Qibin Yang, Jianhua Hao, Bi-functional NaLuF4:Gd31/Yb31/Tm31 nanocrystals: structure controlled synthesis, near-infrared upconversion emission and tunable magnetic properties, J. Mater. Chem. 22 (2012) 9870 9874. [62] A. Xia, et al., Core-shell NaYF4:Yb31,Tm31@FexOy nanocrystals for dual-modality T2-enhanced magnetic resonance and NIR-to-NIR upconversion luminescent imaging of small-animal lymphatic node, Biomaterials 32 (2011) 7200 7208. [63] Z. Liu, et al., Long-circulating Gd2O3:Yb31, Er31 up-conversion nanoprobes as high-performance contrast agents for multi-modality imaging, Biomaterials 34 (2013) 1712 1721. [64] J. Zhou, et al., Fluorine-18-labeled Gd31/Yb31/Er31 co-doped NaYF4 nanophosphors for multimodality PET/MR/UCL imaging, Biomaterials 32 (2011) 1148 1156. [65] Y. Yang, et al., Hydrothermal synthesis of NaLuF4:153Sm,Yb,Tm nanoparticles and their application in dual-modality upconversion luminescence and SPECT bioimaging, Biomaterials 34 (2013) 774 783. [66] J. Xu, et al., Highly emissive dye-sensitized upconversion nanostructure for dual-photosensitizer photodynamic therapy and bioimaging, ACS Nano 11 (2017) 4133 4144. [67] Y. Dai, et al., Doxorubicin conjugated NaYF4:Yb31/Tm31 nanoparticles for therapy and sensing of drug delivery by luminescence resonance energy transfer, Biomaterials 33 (2012) 8704 8713. [68] N.C. Fan, F.Y. Cheng, J.A. Ho, C.S. Yeh, Photocontrolled targeted drug delivery: photocaged biologically active folic acid as a light-responsive tumor-targeting molecule, Angew. Chem. Int. Ed 51 (2012) 8806 8810. [69] B. Yan, J.C. Boyer, D. Habault, N.R. Branda, Y. Zhao, Near infrared light triggered release of biomacromolecules from hydrogels loaded with upconversion nanoparticles, J. Am. Chem. Soc. 134 (2012) 16558 16561. [70] Q. Liu, et al., 18F-Labeled magnetic-upconversion nanophosphors via rare-Earth cation-assisted ligand assembly, ACS Nano 5 (2011) 3146 3157. [71] Z. Xu, et al., Facile synthesis of an up-conversion luminescent and mesoporous Gd2O3 : Er31@nSiO2@mSiO2 nanocomposite as a drug carrier, Nanoscale 3 (2011) 661 667. [72] C. Wang, L. Cheng, Z. Liu, Drug delivery with upconversion nanoparticles for multi-functional targeted cancer cell imaging and therapy, Biomaterials 32 (2011) 1110 1120. [73] D.K. Chatterjee, Z. Yong, Upconverting nanoparticles as nanotransducers for photodynamic therapy in cancer cells, Nanomedicine 3 (2008) 73 82.

340

THERANOSTIC BIONANOMATERIALS

[74] P. Zhang, W. Steelant, M. Kumar, M. Scholfield, Versatile photosensitizers for photodynamic therapy at infrared excitation, J. Am. Chem. Soc. 129 (2007) 4526 4527. [75] C. Wang, H. Tao, L. Cheng, Z. Liu, Near-infrared light induced in vivo photodynamic therapy of cancer based on upconversion nanoparticles, Biomaterials 32 (2011) 6145 6154. [76] K. Deisseroth, Optogenetics, Nat. Methods 8 (2011) 26 29. [77] T.I. Kim, et al., Injectable, cellular-scale optoelectronics with applications for wireless optogenetics, Science 340 (2013) 211 216. [78] E. Pastrana, Optogenetics: controlling cell function with light, Nat. Methods 8 (2011) 24 25. [79] K.D. Anikeeva, Upconversion of Light for Use in Optogenetic Methods. United States Patent, PCT/ US2011/059287, 2011. [80] L. He, et al., Near-infrared photoactivatable control of Ca21 signaling and optogenetic immunomodulation, eLife 4 (2015) e10024. [81] N. Feliu, E. Neher, W.J. Parak, Toward an optically controlled brain, Science 359 (2018) 633 634. [82] P.P. Helmut Schäfer, Otmane Zerzouf, Markus Haase, Synthesis and optical properties of KYF4/Yb, Er nanocrystals, and their surface modification with undoped KYF4, Adv. Funct. Mater. 18 (2008) 2913 2918. [83] J. Tian, et al., Intracellular adenosine triphosphate deprivation through lanthanide-doped nanoparticles, J. Am. Chem. Soc. 137 (2015) 6550 6558. [84] R. Abdul Jalil, Y. Zhang, Biocompatibility of silica coated NaYF4 upconversion fluorescent nanocrystals, Biomaterials 29 (2008) 4122 4128. [85] C.H. Lin, et al., Rare-earth nanoparticle-induced cytotoxicity on spatial cognition memory of mouse brain, Chin. Med. J. 130 (2017) 2720 2725. [86] J.P. Chen, et al., Potential clinical risk of inflammation and toxicity from rare-earth nanoparticles in mice, Chin. Med. J. 131 (2018) 1591 1597. [87] Q. Chen, et al., All-inorganic perovskite nanocrystal scintillators, Nature 561 (2018) 88 93. [88] L. Song, et al., Repeatable deep-tissue activation of persistent luminescent nanoparticles by soft X-ray for high sensitivity long-term in vivo bioimaging, Nanoscale 9 (2017) 2718 2722.

15 Biofunctional Magnetic Nanomaterials for Diagnosis, Therapy, and Theranostic Applications Zheyan Qian, Wei Hu, Yue Pan GUANGDONG PROVINCIAL KEY LABORAT ORY OF MALIGNANT TUMOR E PIGENETICS AND GENE RE GULATION, MEDICAL RESEARCH CE NTER, SUN YAT-SEN MEMORIAL HOSPITAL, SUN YAT-SEN UNIVERSITY, GUANGZHOU, P.R. CHINA

15.1 Introduction Currently, the soaring development of nanotechnology has brought about sophisticated innovations and dramatic breakthroughs, giving a great push to advancements in many fields. The integration of nanotechnology and clinical treatment ushers in a most promising research area and exerts a far-reaching impact on the following exploration [1 4]. In the late 1970s, the concept of magnetic nanoparticles (MNPs) appeared. Iron oxides, magnetic compounds, and magnetic alloys are the most important types of MNPs. Attributed to their quantum effects, such as surface effect, size effect, confinement effect, etc., MNPs exhibit a succession of excellent performance in both stability and size-controllability [5 8]. The benign biocompatibility also guarantees MNPs a reasonable prospect in biomedical application. The biomedical application of MNPs in recent years has aroused a great deal of attention. One typical MNPs system of all the well-established ones involved in biomedical applications is iron oxide-based magnetic nanomaterials with extraordinary physicochemical properties [9 11]. It is a milestone and paves the way for further developments in this area. Due to these aforementioned and other undescribed advantages, an abundant variety of multifunctional MNPs have been discovered and engaged in practical medical applications. Another new material, called magnetic hybrid nanomaterials (MHNs), has also triggered an increasingly heated discussion in the scientific community [12]. Its characteristics including combining diverse ingredients into a single entity and achieving multifunctional properties without losing the individual advantages of each constituent. The features of such neoteric materials provide a wide range of attractive potentials in multimodal diagnosis and multiple therapeutic functions. Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00015-8 © 2019 Elsevier Inc. All rights reserved.

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FIGURE 15–1 Scheme illustrating different functions of bionanomaterials.

Cancer, including malignant tumors, has long been regarded as an unfortunate disease posing a serious threat to human life [13]. To address such a disease, the scientific community, and the medical and engineering circle have made fundamental endeavors in realizing more accurate early diagnosis and more effective follow-up treatment. In this chapter, some of the latest literature about biofunctional MHNs related to cancer diagnosis and treatment will be reviewed. In addition to introducing the synthesis and modification of MHNs, the latest researches about multimodal diagnosis based on the magnetic resonance imaging (MRI), including MRI CT, MRI ECT, and other MRI-based imaging technologies are described. Furthermore, the biomedical applications of MHNs in hyperthermia-based therapy are also briefly described (Fig. 15 1).

15.2 Synthesis and Modification of Biofunctional Magnetic Nanomaterials The preliminary requirements that we addressed for all materials and devices used in clinical diagnosis and treatment were both nontoxicity to organisms and benign biocompatibility, followed by the pursuit of their performance. Nowadays, scientists and engineers have poured lots of energy and enthusiasm into studying and modifying many nanoparticles of different sizes and shapes through a great deal of different synthetic methods. In this section, we attempt to summarize the invaluable experience gained through their scientific research fruits.

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15.2.1 Synthesis of Magnetic Nanomaterials Over the last two decades, developing specific design and synthesis of magnetic nanomaterials for their more effective using in biomedical applications is an attractive direction pursued by a large number of researchers [11]. Under different conditions, these tailordesigned magnetic nanomaterials usually have different morphologies by integrating MNPs with different functional components. Iron oxide-based magnetic nanomaterials, as one type of important multifunctional MHN, are able to prepare a multimodal imaging nanoplatform with full function and adjustable size for tumor treatment. These MHNs are usually synthesized by facile methods from (1) magnetic metals and their oxides, and (2) the transitionmetal-doped oxides and their metal alloys. Owing to their nontoxicity and multifunctionality, MHNs show many promising possibilities in biomedical aspects. The Food and Drug Administration and the European Medicines Agency have approved MHNs.

15.2.2 Hybridization of Magnetic Nanoparticles with Different Morphologies Recently, researchers have tried to synthesize MHNs with different properties in a variety of ways, including using both chemical and physical methods. In addition, the microbial technique is another vital method of MHN synthesis. Compared to other methods, lower cost and higher yield make the method based on chemical synthesis much more widely accepted. Some of the most common chemical methods include the coprecipitation method, in situ synthesis method, sol gel method [14], hydrothermal synthesis method, pyrolysis method, etc. In general, the coprecipitation method can be regarded as the simplest and most effective method for the synthesis of MHNs, but one obvious weakness is that the distribution size of the prepared nanoparticles may not be very uniform. The microemulsion method forms a thermodynamically stable isotropic dispersion by means of interactions between an insoluble two-phase material (such as water and oil) and surfactants. In the mixed solution, the hydrophobic segment of the monolayer surfactant molecules dissolves in the organic phase, while the other end of the hydrophilic phase tends to dissolve in the aqueous phase. Nanoparticles of different sizes and morphologies can be synthesized by regulating the amount of surfactants and cosurfactants, the kinds of organic phases, and the reaction conditions in this two-phase system. The hydrothermal method is another choice to realize the controllability of size and morphology of nanoparticles with great capability of crystallinity. However, due to the high temperature and pressure required for the reaction, this method is not suitable for production on a large scale. Miao’s group synthesized pristine Fe3O4 NPs by the hydrothermal method and pioneered the use of DNA-modified Fe3O4@Au MNPs as selective electrochemical probes for simultaneous biosensing of heavy metal ions [15]. Metal-mediated base pairs formed by C C and T T mismatches in DNA duplexes are the basis for selective recognition. In square wave voltammetry, the stable complex of C-Ag1-C and T-Hg21-T targeted, respectively, by ferrocene (Fc) and methylene blue, showed significant current peaks. This method did not require any amplification processes, thus, its

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practical utility was promising. Jia’s group studied PDA@Fe3O4 obtained via the solvothermal reduction method, which can be used for rapid detection of Staphylococcus aureus [16]. A number of other new synthetic methods are also being developed, such as the microwave ultrasonic method, one-pot method, and seed-based method. Heterogeneity and shell core type are the main morphologies of MHNs, in addition to strawberry type, spherical porous shape, etc. Unique structures also bring about unique properties. For the MHNs with shell-core structure, the surface of the nanoparticles in the core is coated by a plurality of nanofilms. Core and shell connect with each other by physical or chemical action. Therefore, the shell can play a protective role for the substance in the inner core. For example, when iron is characterized as a core, the shell can effectively prevent the oxidation of iron, which effectively prevents denaturation and improves the service life of the product. When it comes to the application of shell core MHNs in the biomedical field, protecting the drug and improving their targeting are of considerable significance when designed as drug-carriers. By the same token, shell core MHNs can decline toxic side effects of drugs by coating biocompatible film nanomaterials. For another MHN of spherical porous shape, due to its unique porous nature, researchers can complex multifunctional molecules on this structure to achieve various specific therapeutic effects. Pan et al. advanced research on the structure of nanoparticles. The dumbbell-shaped MHNs and heterodimer MHNs, as two novel types of nanometer materials, also have proven capabilities of being combined with various functional units to achieve rewarding biocompatibility and noncytotoxic multifunctional parts due to their special structure [17]. Hu’s group focused on the study of MHNs with a strawberry-like structure [18]. In the course of their experiment, they utilized the coprecipitation method, realized the modification of mercaptosuccinic acid to synthesize strawberry-like Fe3O4 Au NPs, and carried out a series of assays about its in vitro cytotoxicity and imaging performance. As reported, this structure equipped with superfine sized Au (B1.2 nm) was verified as being capable of remarkably improving the magnetic properties of the agents and enhancing contrast imaging at atmospheric temperature. Attributed to its excellent performance as a CT-MRI dual-mode contrast agent, this outcome was expected to be an appropriate noninvasive approach for the diagnosis of liver lesions in preference to liver biopsy. Miao’s group discussed the bright prospect of Fe3O4@Ag nanocomposites in the analysis of hydrogen peroxide (H2O2) in biological systems for its advantages of highly sensitivity and selectivity [19]. The porous structure of the core Fe3O4 nanoparticles provided not only a huge specific surface, but also a physical 3D space. Therefore, the loading of Ag nanoparticles was significantly enhanced. Due to the adsorption abilities of the electrode with magnetic properties, the formed Fe3O4@Ag nanocomposites were able to grow on the surface of electrodes. Meanwhile, a highly characteristic silver stripping current can be recorded due to the loaded Ag nanoparticles. However, Ag nanoparticles would be oxidized and dissolved by H2O2 and the transfer of electrons generates electrical signals in this process. After analyzing decreased silver stripping peak current, the level of H2O2 can be revealed.

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15.3 Magnetic Resonance Imaging-Based Multimodal Diagnosis One of the key explanations as to why many cancers are terminal is because they are difficult to diagnose at an early stage and, in turn, the vast majority of patients miss the best treatment period. Thus, the essential point in the process of defeating cancer is to accurately localize the tumor under the guidance of imaging and to then fulfill effective treatments [20]. Herein, early diagnosis, intraoperative localization, and postoperative imaging examination are of great significance in formulating treatment strategies for cancer. Mainly using MRI [21], computed tomography (CT) [22], single-photon emission CT (SPECT), photoacoustic (PA), and positron emission tomography, scientists have endeavored to develop noninvasive imaging techniques and provide imaging of higher resolution and greater comprehensiveness for the diagnosis of various diseases enabling clinical treatment. MRI is an evolving technique for the diagnosis of various diseases, which efficaciously utilizes the interaction between an external magnetic field and an object to offer multiparameter imaging without causing ionizing radiation harm to patients. The principle of MRI is to excite the transition between the energy levels of the hydrogen protons in the human body by applying an external magnetic field, and reconstructing the information of the human body according to the set mathematical program through the magnetic resonance phenomenon. However, each imaging method has its own unique advantages and limitations, including MRI. The main problems with MRI include the low-contrast sensitivity, long scanning time, and high expense. Works to improve the status quo are urgently needed. As shown by various experiments, compared to a traditional single-mode imaging, multimodal imaging has more attractive application prospects because it can combine the advantages of different imaging modes and overcome the respective defects in the existing technology [23 24]. For all these reasons, researches related to multimodal diagnosis based on MRI have aroused a great deal of interest in scientific circles in recent years. Here we present an overview of three different main kinds of multimodal diagnosis.

15.3.1 Magnetic Resonance Imaging Computed Tomography Bimodal Diagnosis As an important medical imaging method for detecting human diseases, CT has been able to obtain high-resolution soft-tissue images without high power and observe high-density structures with X-ray after a long period of development. Despite this, scientists are continuing to pursue a clearer and more comprehensive imaging diagnosis assay, such as the aforementioned multimodal diagnosis. As a matter of fact, MRI CT bimodal diagnosis is one of the most typical and common combinations in bimodal imaging. As shown by studies, such an imaging combination can effectively improve diagnostic accuracy. Many research results have been obtained in this direction. Recent researches made by Pan and co-workers (Fig. 15 2A) show the promising applications of Au Fe3O4 heterostructured nanoparticles in MRI CT bimodal diagnosis [17].

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FIGURE 15–2 (A) An illustration of the Au Fe3O4 heterostructured nanoparticles for MRI CT bimodal imaging diagnosis. (B) An illustration of the application of Fe3O4 Ag125I heterodimers as imaging agents in MRI SPECT bimodal diagnosis. CT, Computed tomography; MRI, magnetic resonance imaging; SPECT, single-photon emission computed tomography. Reprinted with permission from (A) J. Zhu, Y. Lu, Y. Li, J. Jiang, L. Cheng, Z. Liu, et al., Nanoscale 6 (2014) 199 202 © 2014 the Royal Society of Chemistry; (B) J. Zhu, B. Zhang, J. Tian, J. Wang, Y. Chong, X. Wang, et al., Nanoscale 7 (2015) 3392 3395 © 2015 the Royal Society of Chemistry.

Today, Fe3O4 and Au nanoparticles, owing to their excellent biocompatibility, are widely studied and applied in the medical field [2,26]. After the decomposition of Fe(CO)5 coated on the surface of Au nanoparticles, the materials are exposed to air for oxidation to synthesize the Au Fe3O4 heterostructured nanoparticles. Then the tetramethylammonium hydroxide (TMAOH) is used to modify the compounds and obtain water-soluble Au Fe3O4 nanoparticles. Besides successfully verifying the effectiveness of such Au Fe3O4 nanoparticles for in vitro MRI/CT images, these materials also attempted to check the contrast effect in the main organ for testing the feasibility of in vivo imaging in animal trials. This was the first time that Au-based heterostructured nanoparticles as CT imaging agents were tested on a rabbit ventricular structure. The experimental results show that the Au Fe3O4 heterostructured nanoparticles have a comparative enhancement effect on MRI/CT dual-mode imaging. In addition, Pan and co-workers also measured MRI and CT relaxivity to evaluate the feasibility of another nanocomposite, ION@Bi2S3 core shell nanocomposites, as an MRI CT dualmodal agent [27]. The reagent was synthesized by adding Fe(CO)5 to the mixed solution of 1-octadecene (ODE) and oleylamine (OAm) at 180 C. After holding at 180 degrees for 30 minutes, cool the mixture to room temperature. Then centrifuge it and wash it with hexane. A quantity of sulfur is then dissolved into the solution to form iron oxide@sulfur core shell nanoparticles and bismuth trichloride is dispersed evenly into the compound to create the final product. After such a succession of complex advance preparation steps, the final step was the surface modification of materials with mPEG-LA polymer.

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There remains a great deal of frontier research in this field. Feng et al. developed pEGFRtargeted Ba2GdF7 NPs, which had proven to have ideal biocompatibility and easy clearance in the kidney, for MRI CT imaging nanoprobes [28]. Liang et al. studied the feasibility of using Cys-coated FePt NPs as a dual MRI CT contrast medium for glioma therapy [29]. FePt NPs are a potential research object in biomedicine on account of their excellent magnetic effect and chemical stability. The surface coatings, to a great extent, influence the inhibitory effect of FePt NPs on glioma cell proliferation. In this study, the researchers added water-soluble L-cysteine as a coating to improve the biological capacity of the material as a whole and at the same time prevent unnecessary introduction of coatings that may cause damage to the brain. Liu’s group attempted to combine the applications of an MRI CT imaging agent and therapeutic tools in a new synthetic lipid-AuNPs@PDA nanohybrid [30]. This hybrid structure was designed to be a shell core structure. AuNPs@PDA were used as the core. Indocyanine green (ICG) was absorbed onto the surface of the core to form ICG-AuNPs@PDA via electrostatic adsorption. Then, by means of self-assembly, lactobionic acid and other substances functionalized the AuNPs@PDA to make it a targeted therapeutic agent for hepatocellular carcinoma. Incorporating ICG into nanoparticles not only increased the utilization of the material as a photothermal reagent, but also overcame the limitation of ICG on ablation of malignant tumors due to the poor spatial resolution under an aquatic environment. All of these works thoroughly show the infinite possibilities of MHNs for future MRI CT bimodal imaging diagnoses.

15.3.2 Magnetic Resonance Imaging ECT Bimodal Diagnosis As a typical nuclear imaging techniques, ECT has the common advantage of high sensitivity, which means ECT can track quite small amounts of radiolabeled biomolecules in a tiny range. However, ECT is limited by its low resolution and clarity. In view of the successful combination of MRI and CT imaging technologies, the imaging community is actively making attempts at MRI ECT bimodal imaging diagnoses. Pan et al. reported using the bifunctional Fe3O4 Ag125I heterodimers as the MRI ECT dual-modal contrast media to realize the mixed imaging mode [25]. After a facile synthesis of PEGylated Fe3O4 Ag heterostructure nanoparticles, sodium iodine-125 was used to react with the Ag component of the heterostructured nanoparticles and produce the radiolabeled compound (PEGylated Fe3O4 Ag125I heterodimer nanoparticles) at atmospheric temperature. The above compound has been confirmed to have good contrast effects in MRI by observing T2-MRI images of Fe3O4 Ag125I nanoparticles in deionized water at various concentrations. Fig. 15 2B is an illustration of the application in MRI SPECT bimodal imaging of a mouse. By comparing the whole-body SPECT images of mice injected with the heterostructured radionuclide nanoparticle solution via the tail vein with mice injected with Na125I, Pan et al. found that the new compound can be strongly taken up by the liver and spleen and provided a clear SPECT image. All these experiments show the technological feasibility of this material for MRI SPECT dual imaging.

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15.3.3 Magnetic Resonance Imaging-Based Multimodal Diagnosis Sometimes the situation is more complicated, where only using bimodal imaging cannot satisfy the high requirements for a medical diagnosis or show the best imaging diagnosis results. For this reason, a great deal of researchers endeavor to develop trimodal imaging or even combine more imaging techniques, which may be able to meet the needs of more complex conditions. Xu et al. made a successful attempt to synthesize Au cluster-gadolinium oxide integrated nanoparticles via a biomineralization method using bovine serum albumin as the template, which can be used as an optical/CT/MR triple-modal tumor-targeting imaging agent [31]. Au clusters have ideal in view near-infrared fluorescence characteristics (size up to Fermi wavelength) and gadolinium oxide has comparable imaging capability. In addition, because Au clusters have a certain characteristic of easy urination, which effectively promotes rapid renal cleanup and avoids their accumulation, they can also effectively reduce the toxicity of traditional imaging reagents to the human body. The synthetic method using albumin as a template also solves the problem that the chemical conjugation method used in the past was too difficult to operate. Combined with these advantages, optical/CT/MR three-mode imaging agents show great potential. The three-mode imaging ability and potential in vivo toxicity of the particles were also discussed in animal experiments. Fig. 15 3 presents a newly developed tetra-modal imaging agent that can be used to guide combination photothermal and radiotherapeutic therapy [32]. This research was reported by Liu’s group. Bi31 was introduced into FeSe2 nanoparticles at an elevated temperature. The mixture gradually formed FeSe2-dispersed Bi2Se3 nanosheets via cation interchange and was then modified with polyethylene glycol (PEG). A series of subsequent experiments showed that this new composite material could combine the advantages of FeSe2 and Bi2Se3, having the high r2 relaxation rate, excellent X-ray attenuation ability, and

FIGURE 15–3 An illustration of MRI/CT/PA/PET tetra-modal imaging diagnosis and combined treatment using FeSe2-decorated Bi2Se3 nanomaterials which formed as a sheet-like structure. CT, Computed tomography; MRI, magnetic resonance imaging; PET, positron emission tomography. Reprinted with permission from L. Cheng, S. Shen, S. Shi, Y. Yi, X. Wang, G. Song, et al., Adv. Funct. Mater. 26 (2016) 2185 2197. © 2016 John Wiley and Sons.

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strong near-infrared absorption ability. All of these advantages build up a rather favorable foundation for its application in the integration of cancer diagnosis and treatment. At the same time, they also successfully verified the applicability of this material with highly integrated functions to various diagnostic and therapeutic techniques as illustrated in Fig. 15 3.

15.4 Magnetic Nanoparticles for Hyperthermia-Based Therapy Another important point that needs to be improved is the therapeutic technology. Regular cancer treatments, mainly including surgery, photothermal therapy (PTT), chemotherapy, and radiation therapy (RT), still have many drawbacks after a long period of development. For example, the nonspecific killing effect and resistance effect may cause severe toxic side effects to the immune system and destroy surrounding normal tissue. Therefore, it is of great importance to explore new treatment methods or combine the advantages of single treatment methods to realize multimodal collaborative treatment of tumors.

15.4.1 Magnetic Hyperthermia Therapy Hyperthermia therapy has a long history tracing back to the time of Hippocrates, and has been extensively used in the clinical therapy of cancers for quite a long time [33]. The treatment of hyperthermia therapy is anchored in the rising of temperature above body temperature, intensifying the efficacy of the elimination of the targeted cancerous cells. Attributed to its superiority including low biotoxicity and controllability compared to conventional antitumor treatments, hyperthermia therapy has gained much attention and been improved into another reliable and certain treatment means after biological therapies, chemotherapy, radiotherapy and traditional surgery [34]. To realize better treatment, experts have more recently been employing much more advanced heating methods including hot baths, mixed bacterial toxins, infusion heating, high-frequency radialization, magnetic fluid hyperthermia, etc. Currently, plenty of MHNs have been verified as possessing interesting potential which can be used in optimizing the hyperthermia treatment process. Magnetic hyperthermia therapy (MHT) is a therapy exposing the cancerous tissue to an alternating magnetic field. Since magnetic fields cannot be absorbed by living biological tissue, MHT have its effects in vivo deeply without conspicuous side effects. Therefore, it has become a promising research direction. MHNs show fantastic efficiency in converting dispersed magnetic energy into thermal heat as applied to an alternating magnetic field based on both Néel and Brownian relaxation mechanisms [12,35 37] (Fig. 15 4A). Neel relaxation is produced by a random spin flip, a process in which the particles do not rotate, and when the nanoparticles are fixed (e.g., in tumor tissue), it is the only relaxation that leads to magnetic heat generation. The temperature range caused by MHNs influenced by an alternating magnetic field depends on four aspects: the saturation magnetization of the material, the magnetic field intensity, the frequency of the magnetic field direction changes, and the cooling ability of the blood flow in

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FIGURE 15–4 MHNs involved in hyperthermia-based therapy. (A) Néel and Brownian relaxation mechanism. HeLa cell fluorescence microscopy images after MHT with (Zn0.4Mn0 .6)Fe2O4 (left) and Feridex (right), respectively. (B) Diagrammatic illustration of Pt@Fe2O3 nanorods in photo-radio combined therapy. Fluorescence microscopy images of 4T1 cells out of remedy (left) and with Pt@Fe2O3 nanorods under NIR irradiation (right). MHN, Magnetic hybrid nanomaterial; MHT, magnetic hyperthermia therapy; NIR, near-infrared. Reproduced with permission from (A) D. Yoo, J.-H. Lee, T.-H. Shin, J. Cheon, Acc. Chem. Res. 44 (2011) 863 874. © 2011 American Chemical Society; (B) Y. Deng, E. Li, X. Cheng, J. Zhu, S. Lu, C. Ge, et al., Nanoscale 8 (2016) 3895 3899. © 2016 the Royal Society of Chemistry.

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the tumor area. Because of the inducing influences of the magnetic field, the effects will then take on in the lesions and cause damage to the tumor via a noninvasive approach with minimized impact on circumambient tissues. Due to the outstanding magnetic-thermal conversion property and other characteristics, there is broad space and soaring prospects for MHNs to perform well. Nowadays, abundant types of MHNs with diverse structures and different constituents participate actively in this promising application. As reported, Cheon’s group manufactured a type of iron oxide doped with Zn ion, (Zn0.4Mn0.6)Fe2SO4, a sort of nanomaterial being investigated that was equipped with stronger capability in magnetic-thermal energy conversion, better biocompatibility, and weaker biotoxicity compared with the classic Feridex contrast agent [38]. The two fluorescence emission images illustrate the distinctions in vitality of cancerous cells after magnetic hyperthermia treated by the two materials, (Zn0.4Mn0.6)Fe2SO4 (left) and Feridex (right), respectively (Fig. 15 4A). Living cells are labeled by calcein AM (acetoxymethyl) with green emission signals. Obviously, after treatment, the number of living cancerous cells remaining in the left environment is markedly less than in the right, meaning that the Zn dopant nanomaterial performs better than the Feridex agent in MHT, showing excellent application prospects in related fields.

15.4.2 Photothermal Therapy Due to both its noninvasive characteristics and minimal side effects, PTT is considered to be one of the most promising treatments for some cancers. PTT affects tumors by following these steps: (1) injecting materials with high photothermal conversion efficiency into the body; (2) realizing the aggregation of materials in the vicinity of tumor tissue through the application of targeted recognition technology; and (3) converting photon energy into thermal heat through the utilization of external light [usually near-infrared (NIR)] and eliminating cancer cells. Local surface plasmon resonance (LSPR) is the resonance of free electrons on the particle surface caused by incident light. This interaction gives the nanometer photosensitizer unique thermal, optical, and electrical properties. The electrons in the nanoparticles absorb the laser photons and are excited to higher energy levels, where the energy absorbed is converted to heat by electron phonon relaxation. In PTT, MHNs can undergo LSPR to absorb photon energy and convert it into local heat. An NIR (wavelength 650 950 nm) light source is usually advantageous because hemoglobin and water in tissue have low absorption in this waveband, which reduces the damage to living tissue. Therefore PTT can penetrate deep into biological tissue through NIR and destroy deep-tissue lesions. It can be an ideal candidate to combine with other therapies to obtain a better curative effect [40]. Since tumor cells are more temperature-sensitive than normal healthy human tissues, PTT is regarded as a much more effective and comprehensive form of treatment than conventional surgery involving physical elimination [41,42]. Multifunctional MHNs have recently aroused a great deal of attention on account of their secure, controllable, biodegradable, and nontoxic properties when involved in PPT. However, confined by the toxicity and

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biopersistent potential of MHNs, especially in materials such as Au, carbon nanotubes, and graphene, the utilization of MHNs in this clinical field has not gained much ground. The manufacture of both biocompatible and nontoxic MHNs hence is an urgent requirement. For example, PEGylated Fe@Fe3O4 MHNs synthesized by Zhou et al. possess not only high efficiency in photothermal conversion, but also low toxicity and high stability; [43] Li et al. creatively innovated an IONC@Au-PEG nanoplatform presenting high effectiveness in handling the elimination of cancers and enhancing imaging simultaneously without high toxicity [44].

15.4.3 Combined Therapy In practice, the efficacy of combined therapy is usually better than any single therapy. Combined therapy has the potential to utilize the advantages of each therapy involved and even improve these to a new standard. Moreover, in some cases, the drawbacks of each can even be lessened through synergistic therapy [45]. A combination of well-designed treatments will have a synergistic effect on the treatment of tumors, thereby reducing side effects while enhancing the effects of treatment. Combining chemotherapy with heat therapy, for example, produces a synergistic effect that not only increases the effectiveness of cancer treatment but also reduces the dose of anticancer drugs required. Therefore, the development of a biodegradable multifunctional nanoplatform that can combine multiple therapies is of great significance in the field of tumor treatment. With more and more achievements revealed to the public, combined therapy has drawn soaring attention. For instance, Pan and co-workers fabricated Pt@Fe2O3 nanorods that combine both PTT and RT [39] (Fig. 15 4B). What was reported previously was that Pt is a fantastic radiosensitizer which means that the radiation dosage can be limited to a small amount. Since radiation kills cells without distinction, a lower radiation dosage means less unintended damage. Aside from that, iron oxide acts well in PTT, meaning there can be a synergistic effect to minimize the dose of the agent but achieve a similar, or even better, curative effect without huge harm to the body [46]. An experiment has taken place to assess the curing efficacy of Pt@Fe2O3. 4T1 cells are selected as the targets and marked by AM and propidium iodide, and the result, after treatment, is that there is a significant decline in the quantity of target cells (Fig. 15 4B).

15.5 Magnetic Nanoparticles for Theranostic Treatment The timely detection and treatment of cancer is badly hampered by the redundant but inevitable time lapse between diagnosis and treatment, certainly delaying the follow-up treatment. However, the advantageous properties of magnetic nanomaterials make the implementation of hyperthermia possible, providing conditions to handle this dilemma and improve the situation. Nanotechnology can be used to realize the target treatment and diagnosis, reducing the side effects of conventional chemotherapy, and monitoring the disease [47]. Multifunctional MHNs with comprehensive diagnostic and therapeutic functions can be

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broadly used in multimodality imaging and collaborative therapy, both improving diagnostic accuracy and enhancing therapeutic efficacy [48]. Among the noninvasive imaging techniques, MRI is beneficial in spatial resolution without consonant capability in sensitivity, yet CT provides 3D visualization with dissatisfactory resolution [49]. Pan and co-workers, thereupon, fabricated PEGylated Fe@Bi2S3 nanocomposites to combine these two methods together, aiming at reinforcing their merits and avoiding the drawbacks of each method [50]. Since its instinct narrow direct band gap led to great absorption ability, Bi2S3 shows strong NIR absorption, which is a benefit in PTT. In addition, Bi2S3 is an excellent agent used in RT and CT due to its high atomic number. What is more, Fe’s magnetic property has the potential to be utilized in MRI as well. Up to now, experiments both in vitro and in vivo have indicated that Fe@Bi2S3 performs well in tumor treatment and has great biocompatibility (Fig. 15 5A).

FIGURE 15–5 (A) An illustration of multifunctional PEGylated Fe@Bi2S3 nanoparticles for synergistic thermoradiotherapy under the guidance of MRI CT bimodal imaging. (B) Illustration of peptide-Fe3O4@MSNs for the combined process of enzyme-responsive drug delivery, MRI, and chemotherapy. CT, Computed tomography; MRI, magnetic resonance imaging; MSN, mesoporous silica-based nanoplatform. Reprinted with permission from (A) E. Li, X. Cheng, Y. Deng, J. Zhu, X. Xu, P.E. Saw, et al., Biomater. Sci. 6 (2018) 1892 1898 © 2018 the Royal Society of Chemistry; (B) E. Li, Y. Yang, G. Hao, X. Yi, S. Zhang, Y. Pan, et al., Nanotheranostics 2 (2018) 233 242. © 2018 Ivyspring International Publisher.

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Another nanomaterial, multifunctional mesoporous silica-based nanoplatforms (MSNs), has also been investigated and shown to have excellent biocompatibility and high enzyme responsiveness, and has promise in therapeutic applications. Pan et al. devoted huge efforts in setting up the Fe3O4@MSNs-based drug-delivery system (Fig. 15 5B). MMP-2 responded peptide substrate (PLGVR) has been involved in further modification of the Fe3O4@MSNs’ surface. This system can realize the simultaneously process of MRI-guided diagnosis and real-time monitoring of controlled drug release, effectively hindering tumor growth and realizing accurate diagnosis [51].

15.6 Challenges and Conclusions Biofunctional magnetic nanomaterials possess a variety of vital characteristics which contribute to their excellent biofunctional utilities including multimodal diagnosis and theranostic therapy. This chapter briefly portrays the synthesis, modification, some progress, and farreaching achievements correlated to MHNs. In image-guided multimodal diagnosis, MHNs systematically integrate MRI, CT, ECT, and other methods not described in the chapter together to attain a much better effect. What is more, MHNs can be applied in several kinds of therapies such as MHT, PTT, and RT, and all these remedy methods can be legitimately combined to obtain an enhanced synergistic effect with excellent several-fold efficacy. Moreover, the diagnosis and the treatment even have the capacity to combine with each other with the existence of MHNs. Theranostic treatment realizes the simultaneity of monitoring and treatment to fully meet the requirements of patients, doctors, and the scientific community. Even though this unprecedented era has been credited to both the booming nanotechnology and to advanced scientific research that have emerged, there is no denying that there remains a long way to go. First and foremost, correlated researches into most MHNs in biomedical applications have only been carried out in animal experiments or in vitro, with challenges remaining before MHNs can be put into practical production and application on a large scale. Second, MHNs remain relatively large, which is not good for their utilization in vivo. Third, further optimization is required in their photothermal response ability. However, any potential flinch when facing the challenges listed is antithetical to the attitude that the chapter wants to deliver. What is really required is more critical progress in optimizing the material system and the enterprising spirit. The authors sincerely hope that MHNs will play an increasing critical role in cancer diagnosis and treatments in the not distant future.

Acknowledgments This work was supported by the National Key R&D program of China (2018YFB1105700), National Natural Science Foundation of China (51402203), and Guangdong Science and Technology Department (2017B030314026).

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References [1] N.L. Rosi, C.A. Mirkin, Chem. Rev. 105 (2005) 1547 1562. [2] L. Cheng, C. Wang, L. Feng, K. Yang, Z. Liu, Chem. Rev. 114 (2014) 10869 10939. [3] J. Xie, G. Liu, H.S. Eden, H. Ai, X. Chen, Acc. Chem. Res. 44 (2011) 883 892. [4] Y. Gao, Y. Liu, C. Xu, Magnetic Nanoparticles for Biomedical Applications: From Diagnosis to Treatment to Regeneration, Springer, London, 2014. [5] K.E. Sapsford, W.R. Algar, L. Berti, K.B. Gemmill, B.J. Casey, E. Oh, et al., Chem. Rev. 113 (2013) 1904 2074. [6] L. Wu, A. Mendozagarcia, Q. Li, S. Sun, Chem. Rev. 116 (2016) 10473. [7] J. Gao, X. Chen, Z. Zhao, Octapod Iron Oxide Nanoparticles As High-Performance T2 Contrast Agents For Magnetic Resonance Imaging, 2016, 222 228. [8] N. Lee, T. Hyeon, Chem. Soc. Rev. 43 (2012) 2575 2589. [9] Y. Hu, S. Mignani, J.-P. Majoral, M. Shen, X. Shi, Chem. Soc. Rev. 47 (2018) 1874 1900. [10] M. Colombo, S. Carregal-Romero, M.F. Casula, L. Gutiérrez, M.P. Morales, I.B. Böhm, et al., Chem. Soc. Rev. 41 (2012) 4306 4334. [11] A.K. Gupta, M. Gupta, Biomaterials 26 (2005) 3995 4021. [12] X. Tian, L. Zhang, M. Yang, L. Bai, Y. Dai, Z. Yu, et al., Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 10 (2018) e1476. [13] R. Siegel, J. Ma, Z. Zou, A. Jemal, CA-Cancer J. Clin. 64 (2014) 9 29. [14] J. Xu, H. Yang, W. Fu, K. Du, Y. Sui, J. Chen, et al., J. Magn. Magn. Mater 309 (2007) 307 311. [15] P. Miao, Y. Tang, L. Wang, ACS Appl. Mater. Interfaces 9 (2017) 3940 3947. [16] X. Gao, X. Yao, Z. Zhong, L. Jia, Talanta 186 (2018) 147 153. [17] J. Zhu, Y. Lu, Y. Li, J. Jiang, L. Cheng, Z. Liu, et al., Nanoscale 6 (2014) 199 202. [18] H.Y. Zhao, S. Liu, J. He, C.C. Pan, H. Li, Z.Y. Zhou, et al., Biomaterials 51 (2015) 194 207. [19] Z. Guo, J. Xu, J. Zhang, Y. Hu, Y. Pan, P. Miao, ACS Appl. Biol. Mater. 1 (2018) 367 373. [20] M.F. Kircher, U. Mahmood, R.S. King, R. Weissleder, L. Josephson, Cancer Res. 63 (2003) 8122. [21] Z. Shen, A. Wu, X. Chen, Mol. Pharm 14 (2017) 1352 1364. [22] D. Huo, J. He, H. Li, H. Yu, T. Shi, Y. Feng, et al., Colloids Surf., B 117 (2014) 29 35. [23] X. Li, C. Wang, H. Tan, L. Cheng, G. Liu, Y. Yang, et al., Biomaterials 108 (2016) 71 80. [24] A. Louie, Chem. Rev. 110 (2010) 3146 3195. [25] J. Zhu, B. Zhang, J. Tian, J. Wang, Y. Chong, X. Wang, et al., Nanoscale 7 (2015) 3392 3395. [26] J. Chen, D. Wang, J. Xi, L. Au, A. Siekkinen, A. Warsen, et al., Nano Lett. 7 (2007) 1318 1322. [27] J. Zhu, J. Wang, X. Wang, J. Zhu, Y. Yang, J. Tian, et al., J. Mater. Chem. B3 (2015) 6905 6910. [28] Y. Feng, H. Chen, B. Shao, S. Zhao, Z. Wang, H. You, ACS Appl. Mater. Interfaces 10 (2018) 25511 25518. [29] S. Liang, Q. Zhou, M. Wang, Y. Zhu, Q. Wu, X. Yang, Int. J. Nanomed. 10 (2015) 2325 2333. [30] Y. Zeng, D. Zhang, M. Wu, Y. Liu, X. Zhang, L. Li, et al., ACS Appl. Mater. Interfaces 6 (2014) 14266 14277. [31] C. Xu, Y. Wang, C. Zhang, Y. Jia, Y. Luo, X. Gao, Nanoscale 9 (2017) 4620 4628. [32] L. Cheng, S. Shen, S. Shi, Y. Yi, X. Wang, G. Song, et al., Adv. Funct. Mater. 26 (2016) 2185 2197.

356

THERANOSTIC BIONANOMATERIALS

[33] M. Soleymani, M. Edrissi, A.M. Alizadeh, J. Mater. Chem. B 5 (2017) 4705 4712. [34] C.S.S.R. Kumar, F. Mohammad, Adv. Drug. Deliv. Rev. 63 (2011) 789 808. [35] L. Lartigue, P. Hugounenq, D. Alloyeau, S.P. Clarke, M. Lévy, J.-C. Bacri, et al., ACS Nano 6 (2012) 10935 10949. [36] S.-H. Noh, W. Na, J.-T. Jang, J.-H. Lee, E.J. Lee, S.H. Moon, et al., Nano Lett. 12 (2012) 3716 3721. [37] D. Yoo, J.-H. Lee, T.-H. Shin, J. Cheon, Acc. Chem. Res 44 (2011) 863 874. [38] J.-T. Jang, H. Nah, J.-H. Lee, S.H. Moon, M.G. Kim, J. Cheon, Angew. Chem. Int. Ed. 48 (2009) 1234 1238. [39] Y. Deng, E. Li, X. Cheng, J. Zhu, S. Lu, C. Ge, et al., Nanoscale 8 (2016) 3895 3899. [40] Q. Guan, Y. Dai, Y. Yang, X. Bi, Z. Wen, Y. Pan, Nano Energy 51 (2018) 333 339. [41] S. Shen, S. Wang, R. Zheng, X. Zhu, X. Jiang, D. Fu, et al., Biomaterials 39 (2015) 67 74. [42] M. Saeed, W. Ren, A. Wu, Biomater. Sci. 6 (2018) 708 725. [43] P. Zhou, H. Zhao, Q. Wang, Z. Zhou, J. Wang, G. Deng, et al., Adv. Healthcare Mater. 7 (2018) 1701201. [44] Z. Li, S. Yin, L. Cheng, K. Yang, Y. Li, Z. Liu, Adv. Funct. Mater. 24 (2013) 2312 2321. [45] X. Zhou, X. Lv, W. Zhao, T. Zhou, S. Zhang, Z. Shi, et al., RSC Adv. 8 (2018) 18647 18655. [46] E. Porcel, S. Liehn, H. Remita, N. Usami, K. Kobayashi, Y. Furusawa, et al., Nanotechnology 21 (2010) 85103. [47] J. Wei, X. Shuai, R. Wang, X. He, Y. Li, M. Ding, et al., Biomaterials 145 (2017) 138 153. [48] J. Chen, M. Li, X. Yi, Q. Zhao, L. Chen, C. Yang, et al., Part. Part. Syst. Char. 34 (2017) 1600330. [49] Y. Gao, J. Lim, S.-H. Teoh, C. Xu, Chem. Soc. Rev. 44 (2015) 6306 6329. [50] E. Li, X. Cheng, Y. Deng, J. Zhu, X. Xu, P.E. Saw, et al., Biomater. Sci. 6 (2018) 1892 1898. [51] E. Li, Y. Yang, G. Hao, X. Yi, S. Zhang, Y. Pan, et al., Nanotheranostics 2 (2018) 233 242.

16 AIEgen-Based Fluorescent Nanoparticles for Long-Term Cell Tracing Hui Gao, Sijie Chen MING WAI LAU CE NTRE FOR REPARATIVE MEDICI NE, KAROLINSKA INSTITUTET, HONG KONG, P.R. CHINA

16.1 Introduction Cells are the smallest unit of life. They are not always steady but they are dynamic in the human body [1]. Cell behavior, such as cell migration, plays an important role in normal biological and pathological processes [2]. Fibroblasts and stem cells migrate during wound healing [2]. Cancer metastasis, the spreading of cancer cells, is the main cause of cancer death [3]. Noninvasive cell tracking enables long-term and prompt monitoring of cell behavior, which allows researchers and medicinal practitioners to understand various complex biological processes, study disease progression dynamics, and develop disease treatment strategies [4,5]. Current primary imaging techniques include optical imaging [such as fluorescence (FL) imaging], magnetic resonance imaging (MRI), radionuclide imaging, computed tomography (CT) imaging, positron emission tomography, photoacoustic (PA) imaging, etc. [6 8]. Among these imaging techniques, FL imaging has received much attention due to the ease of access, superior imaging sensitivity, high spatial resolution, and absence of radiationrelated risks. Therefore FL imaging techniques have been widely used for cell tracing. Typical fluorescent cellular tracers include fluorescent proteins (FPs), small-molecule fluorescent dyes, and fluorescent nanoparticles (NPs) [9]. Expression of green fluorescent protein (GFP) or its variants via viral transduction or nonviral plasmid transfection is commonly used for cell tracking [4]. However, the labeling efficiency of this method is dependent on the FP transfection efficiency, which is cell-type dependent. In addition, its safety concerns are still not fully addressed due to the introduction of random insertional mutations at integration sites. Cell labeling by a fluorescent agent is fairly straightforward, simple, low-cost and does not involve genetic modification of the cells. As a result, it has also been investigated intensively. Quantum dots (QDs) (e.g., CdSe, PbS), the highly luminescent inorganic NPs, are resistant to photobleaching but are inherently toxic to cells [10]. In comparison, Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00016-X © 2019 Elsevier Inc. All rights reserved.

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organic molecules are rich in variety and more compatible with living cells, but many of themsuffer from small Stokes shifts, low photostability, and high cell leakage rate. Therefore it is not easy to achieve long-term cell tracing by small organic fluorophores. By incorporating organic fluorophores into NPs, the resultants, organic dye-based NPs, can achieve longer cellular retention time, lower exocytosis rate, and better stability than their discrete molecular counterparts [11]. However, the development of these bright organic dye-based NPs is hampered by the aggregation-caused quenching (ACQ) effect, which is a common problem for most conventional organic fluorophores (Fig. 16 1A): fluorophores in close proximity quench themselves. To avoid self-quenching, only limited amount of dye molecules can be doped into one NP. The discovery of organic fluorogens with aggregation-induced emission (AIE) characteristics opens a new avenue for the development of organic dye-based NPs. Different from the ACQ dyes, AIE molecules (AIEgens) are weak emitters when dissolved in solvent but start to emit strongly when aggregates are formed (Fig. 16 1B). They are bright solid-state emitters. Detailed mechanistic studies demonstrated that the AIE phenomenon is attributed to the restriction of intramolecular motion and the prevention of possible π π interactions due to the nonplanar conformations of AIE molecules. The unique features of AIEgens suggest that they have a great potential for fabricating highly emissive fluorescent NPs. Various organic AIEgen-based NPs with high fluorescence, excellent photostability, great cellular retention, and good biocompatibility have been developed in recent years [11]. AIEgen-based fluorescent NPs have been applied in many theranostics applications, including bioimaging, cell tracking, vascular disease diagnosis, image-guided surgery, and image-guided therapy. In this chapter, we are going to introduce AIEgen-based NPs and their application in long-term cell tracking.

FIGURE 16–1 Typical examples of (A) conventional fluorophores with ACQ problem and (B) fluorophores with AIE property [1]. ACQ, aggregation-caused quenching; AIE, aggregation-induced emission. Reproduced from S. Chen, H. Wang, Y. Hong, B.Z. Tang, Fabrication of fluorescent nanoparticles based on AIE luminogens (AIE dots) and their applications in bioimaging, Mater. Horiz. 3 (4) (2016) 283 293 with permission. © 2016 Royal Society of Chemistry.

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16.2 Fabrication of AIEgen-Based Nanoparticles 16.2.1 AIEgens Various AIEgens have been designed and developed in the past few years. Their emission colors range from blue to red, and even near-infrared. A few typical examples of AIEgens are listed in Table 16 1 [11 14]. Tetraphenylethene (TPE) is a classic AIEgen that emits blue light under ultraviolet (UV) light irradiation (Fig. 16 1B, Table 16 1). By introducing electron-donating or -withdrawing groups to TPE moieties, the newly formed AIEgens can emit light in the green, yellow, or red regions. For example, adding a benzothiazolium unit to a TPE molecule generates a hemicyanine dye, whose solid state emission color can be tuned from yellow, orange, to red by controlling the transformation between the crystalline and the amorphous state [15]. Another example is 4,7-bis[4-(1,2,2-triphenylvinyl)phenyl]benzo-2,1,3-thiadiazole (BTPETD), which has two TPE units linked by a 2,1,3-thiadiazole (TD) unit. Its emission color is green (Table 16 1) [13]. When linking two TPE units and one TD unit with thiophene rings, the resultant AIEgen (BTPEBTTD) shows a large red-shift in the fluorescence emission compared with BTPETD. The two thiophene rings can relieve the steric effect imposed by the two TPE units on the TD core [13]. The bigger space between two TPE units promotes the BTPEBTTD molecules to stack in a cross-like pattern, which results in stronger intermolecular interactions. Hence, a red shift in the FL spectrum of BTPEBTTD compared with BTPETD is observed. Similarly, by linking TPE, triphenylamine, and fumaronitrile together, 2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)phenyl)fumaronitrile (TPETPAFN) is formed, and it displays red fluorescence (Table 16 1). All these AIEgens are usually soluble in common solvents [tetrahydrofuran (THF), DMSO, etc.] and have a high quantum yield (QY) (up to 100%) in aggregate or solid state. As shown in Table 16 1, BTPETD reaches a QY of 89% in powder form [13]. It is worth mentioning that many AIEgens are biocompatible and are suitable for various applications, including cell imaging, photothermal therapy, photodynamic therapy, drug release monitoring, etc. [1,16]. AIEgens with large Stokes shifts ($100 nm) can minimize self-absorption, making them favorable candidates for bioimaging. In recent years, a number of AIEgens have been found or have been designed as efficient photosensitizers. Some exhibit better photosensitization performance than many reported commercial systems. Such AIEgens are excellent photosensitizer materials for image-guided photodynamic therapy application. Although not covered in this chapter, readers interested in this topic are referred to Ref. [16].

16.2.2 Introduce AIEgens Into Nanoparticles 16.2.2.1 Noncovalent Binding Method AIEgen-based NPs can be synthesized via common NP preparation methods through noncovalent binding. Some of the hydrophobic AIEgens can self-assemble into nano-aggregates. For example, silole-N nano-aggregates were formed spontaneously by injecting THF dissolved silole-N into water [7]. Similarly, the 2-(2-(4-(1,2,2-triphenylvinyl)phenyl)-4H-chromen-4-ylidene)malononitrile (TPE-FN) amorphous aggregates were obtained by adding

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Table 16–1 AIEgens

Typical AIEgens: Molecular Structures and Optical Properties Molecular Structure

Absorption, FL Spectrum, QY, фF

TPE [12]

BTPETD [11,13]

TPETPAFN [14]

BTPETD, 4,7-bis[4-(1,2,2-triphenylvinyl)phenyl]benzo-2,1,3-thiadiazole; FL, fluorescence; QY, quantum yield; TPE, tetraphenylethene; TPETPAFN, 2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)phenyl)fumaronitrile. Source: Reproduced from S. Chen, H. Wang, Y. Hong, B.Z. Tang, Fabrication of fluorescent nanoparticles based on AIE luminogens (AIE dots) and their applications in bioimaging, Mater. Horiz. 3 (4) (2016) 283 293; G. Feng, C.Y. Tay, Q.X. Chui, R. Liu, N. Tomczak, J. Liu, et al., Ultrabright organic dots with aggregation-induced emission characteristics for cell tracking, Biomaterials 35 (30) (2014) 8669 8677; Z. Zhao, S. Chen, J.W.Y. Lam, C.K.W. Jim, C.Y.K. Chan, Z. Wang, et al., Steric hindrance, electronic communication, and energy transfer in the photo- and electroluminescence processes of aggregation-induced emission luminogens, J. Phys. Chem. C 114 (17) (2010) 7963 7972; Z. Zhao, C. Deng, S. Chen, J.W.Y. Lam, W. Qin, P. Lu, et al., Full emission color tuning in luminogens constructed from tetraphenylethene, benzo-2,1,3-thiadiazole and thiophene building blocks, Chem. Commun. 47 (31) (2011) 8847 8849; K. Li, W. Qin, D. Ding, N. Tomczak, J. Geng, R. Liu, et al., Photostable fluorescent organic dots with aggregation-induced emission (AIE dots) for noninvasive long-term cell tracing, Sci. Rep. 3 (2013) 1150 with permission. © 2016 Royal Society of Chemistry, 2014 Elsevier, 2010 American Chemical Society, 2011 Royal Society of Chemistry, and 2013 Nature, respectively.

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TPE-FN/THF solution into DI-H2O under ultrasonication, and the aggregates were around 500 nm in diameter (Fig. 16 2A) [17]. Despite the simplicity of this method, it cannot be used for AIEgens that are too hydrophobic, for example, TPETPAFN (an adduct of TPE, triphenylamine, and fumaronitrile; Table 16 1) [1]. Such AIEgens prefer to form large

FIGURE 16–2 Examples of AIEgen-based NPs [16]. (A) Amorphous TPE-FN nanoaggregates [17]; (B) lipid-PEGencapsulated AIE NPs [1,18]; (C) AIE-BSA NPs [19]; (D) AIE-SiO2 NPs [1,20]; (E) TPE-FN nanocrystals [17]; (F) CH3NH3PbBr3-AIE nanocrystals [21]; (G) AIE-chitosan NPs [1,22]; (H) photo-crosslinked AIE-OXE NPs [23]. AIE, aggregation-induced emission; BSA, bovine serum albumin; NP, nanoparticle; OXE, oxetane; PEG, poly(ethylene glycol); TPE, tetraphenylethene; TPE-FN, 2-(2-(4-(1,2,2-triphenylvinyl)phenyl)-4H-chromen-4-ylidene)malononitrile. Reproduced from S. Chen, H. Wang, Y. Hong, B.Z. Tang, Fabrication of fluorescent nanoparticles based on AIE luminogens (AIE dots) and their applications in bioimaging, Mater. Horiz. 3 (4) (2016) 283 293; H. Gao, X. Zhao, S. Chen, AIEgen-based fluorescent nanomaterials: fabrication and biological applications, Molecules 23 (2) (2018) 419; X. Fang, X. Chen, R. Li, Z. Liu, H. Chen, Z. Sun, et al., Multicolor photo-crosslinkable AIEgens toward compact nanodots for subcellular imaging and STED nanoscopy, Small 13 (41) (2017) 1702128 with permission. © 2016 Royal Society of Chemistry, 2018 MDPI, 2017 Wiley, 2012 Royal Society of Chemistry, 2012 Wiley, 2016 American Chemical Society, 2017 Royal Society of Chemistry, 2016 Elsevier, and 2017 Wiley, respectively.

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precipitates in solutions with high water content. Hence, amphiphilic molecules are used to stabilize and encapsulate the hydrophobic AIEgens into NPs via nanoprecipitation. A typical protocol involves injecting the mixture of hydrophobic AIEgens, amphiphilic molecules, and organic solvent into DI-H2O under sonication or continuous stirring. The hydrophobic parts of amphiphilic molecules would entrap lipophilic AIEgens together as the core, while the hydrophilic ends would be exposed to water and form a shell to stabilize the NPs (Fig. 16 2B). To date, various AIEgen-based NPs have been fabricated in this way. Examples of different AIEgen-based NPs synthesized by such a method can be found in Refs. [10,24 26]. AIEgens can also be incorporated into polymeric or inorganic matrices via nanoprecipitation method. For example, AIEgens (e.g., BTPETD or TPETPAFN) can assemble together with amphiphilic polymers (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(poly(ethylene glycol))-2000] (DSPE PEG2000)) to form NPs simply by pouring the mixture of the two in organic solvent into water [11,13]. The desolvation technique of incorporating AIEgens into bovine serum albumin (BSA) matrices was also used [19,27]. TPE-EPA-DCM-based BSA composite NPs with a diameter of approximately 100 nm were formed via a precipitation method assisted by desolvation technique (Fig. 16 2C). In addition, inorganic materials were also employed as matrices to load AIEgens via noncovalent binding methods [20,28,29]. A typical example is the incorporation of AIEgen into silica NPs via sol gel reaction using tetraethyl orthosilicate (TEOS) in the presence of TPE molecules (Fig. 16 2D) [20]. The above-mentioned examples of AIEgen-based NPs are all in an amorphous state. Recently, researchers observed that AIE nanocrystals show brighter fluorescence than the corresponding amorphous AIE NPs. A mechanism investigation revealed that crystallization reduces the potential of intramolecular motions, as NPs in a crystallized state are more compact than those in an amorphous state [17,30]. Fateminia et al. [17] chose TPE-FN as a model AIEgen to study the crystallization effects (Fig. 16 2E). The TPE-FN nanocrystals were fabricated through a bottom-up method, in which probe ultrasonication was employed as a mechanical stress source. The TPE-FN crystal seed suspension was prepared by simply injecting TPE-FN/THF solution into THF/DI-H2O solution (fw 5 65%) in a vial, and then the mixture was tightly covered and kept in the dark for a certain time to enable a complete crystallization process. To synthesize TPE-FN nanocrystals, extra TPE-FN molecules were added to DI-H2O while being probe-sonicated, and subsequently, the crystal seed suspension and additional THF solvent were added into the reaction system. The final products were obtained after removing THF via a dialysis process. A property test revealed that the TPE-FN nanocrystals showed brighter fluorescence than the amorphous TPE-FN aggregates, indicating that crystallization with more compact structure can effectively minimize the intramolecular motions of AIEgens. Interestingly, AIEgens with functional ligands have been demonstrated with the ability to assemble semiconductor nanocrystals into superstructures. For example, a TPE derivative, 3-(4-(1,2,2-triphenylvinyl)phenoxy)propan-1-amine, was applied to synthesize CH3NH3PbBr3 nanocrystals (Fig. 16 2F) [21]. The CH3NH3PbBr3 nanocubes with a side length of approximately 11.1 nm packed into well-ordered superstructures, originating from the interactions

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between the AIE molecules. The properties of such superstructures can be tuned by changing the chain length of the TPE derivative. For instance, the chain length can affect the distance between two adjacent nanocubes and consequently results in different fluorescent properties in the formed particles.

16.2.2.2 Covalent Binding Method The advantage of the noncovalent binding approaches is that they require less dye modification. Some other AIEgen-based NPs are fabricated via covalent binding method. The NPs fabricated by such a method show better chemical and physical stability [1]. Compared with noncovalent binding, the loading ratio of the AIEgens in NPs is easier to be controlled via covalent binding. AIEgens can be covalently linked to polymers, forming AIEgen-modified polymer first. AIEgen-based NPs are then fabricated by using these fluorescent polymers as raw materials (Fig. 16 2G and H) [5,31,32]. Examples include AIE-chitosan (CS) NPs and AIE-SiO2 NPs. To prepare TPE-CS NPs, TPE was first attached to CS by the reaction between isothiocyanate (NCS) groups in TPE-NCS and amine groups in CS. Then TPE-CS NPs were fabricated by ionic gelation method by mixing sodium tripolyphosphate (TPP) with TPE-CS. The crosslinking reaction between the positively charged amino groups of CS and the negatively charged phosphate groups of TPP led to the formation of TPE-CS NPs (Fig. 16 2G) [33]. Alternatively, AIEgens with functional groups can be firstly introduced into the NP matrices, and then covalently bind to the matrix materials under proper external stimuli triggering (Fig. 16 2H) [23]. For example, by coprecipitating TPE derivatives functionalized with oxetane groups (AIE-OXE) and polystyrene-OXE, the initial AIE NPs (around 15 nm in diameter) were obtained. Subsequently, the OXE groups in the NPs can be photo-crosslinked by cationic ring opening polymerization upon UV light irradiation, so as to form the final compact and stable AIE NPs. AIEgens can also be embedded in inorganic matrix materials such as silica via covalent binding. Faisal et al. demonstrated that AIE-SiO2 NPs with a core shell structure can be made by sol gel reaction of AIE functionalized siloxanes followed by a second sol gel reaction with tetraethoxysilane [34]. The AIEgens were covalently linked with the silica matrices and showed good stability.

16.2.3 Functionalization of the AIEgen-Based Nanoparticles These AIEgen-based fluorescent NPs can be modified by pre- or post-modification to enable them with specific functions. Several review papers that provide details on the surface modification, functionalization, and applications of AIEgen-based fluorescent NPs are available in Refs. [1,16,31,35].

16.2.3.1 Enhancing Targeting Efficiency The AIEgen-based NPs can be decorated with bioactive targeting molecules, such as peptides, proteins, folic acid (or folate), etc., to enhance their targeting efficiency [1,36,37].

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Folate is a vitamin that is required by all living cells, and many types of cancer cells overexpress folate receptors on the plasma membrane [36]. Hence, folate modification of NPs is widely used by researchers as one of the most popular approaches to target cancer cells. Folate can be easily introduced to the AIE NPs via a prefunctionalization method, i.e., predecorating folate molecules to matrix materials and then nanoprecipitating the folatemodified matrix materials with AIEgens into NPs (Fig. 16 3A). Typical folate-decorated matrix materials include poly[lactide-co-glycolide] PEG folate (PLGA PEG folate), PLGA folate, and lipid PEG folate [18,36,38]. The formed fluorescent NPs can easily target the cells with overexpressed folate receptors. Surface modification of AIEgen-based NPs with targeting molecules can also be achieved by the postmodification method. Biotin is a tumor-targeting vitamin molecule. It is rich in carboxylic acid groups that can react with molecules with amino groups via amidation reaction. Hence, to realize decoration of a biotin, AIEgen-based NPs were modified with amino groups, for example, silole-based AIE-SiO2 NPs shown in Fig. 16 3B, and then reacted with biotin [39]. Similarly, by incorporating AIEgens into encapsulation matrix containing lipidPEG-NH2, the formed AIEgen-based NPs were prefurnished with amino groups, which can react with cell-penetrating peptide HIV-1 transactivator of transcription (Tat) protein via carbodiimide-mediated coupling, yielding Tat AIE NPs (Fig. 16 3C). Such modified AIE NPs showed promising cell targetability.

FIGURE 16–3 Functionalization of AIEgen-based NPs: (A) premodification of the matrix materials; (B) surface modification of the amine-furnished AIE-SiO2 NPs or (C) lipid/polymer-based AIE NPs with targeting groups (e.g., biotin); (D) postmodification to enable dual functionalization of AIE NPs; (E) incorporation of functional component (e.g., magnetic Fe3O4) as the core part of AIE-SiO2 NPs [1]. AIE, aggregation-induced emission; NP, nanoparticle. Reproduced from S. Chen, H. Wang, Y. Hong, B.Z. Tang, Fabrication of fluorescent nanoparticles based on AIE luminogens (AIE dots) and their applications in bioimaging, Mater. Horiz. 3 (4) (2016) 283 293, with permission. © 2016 Royal Society of Chemistry.

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16.2.3.2 Enabling Multifunctionality Different imaging techniques have different advantages and limitations [40 42]. For example, optical imaging possesses high sensitivity with optical molecular probes, but cannot be used for deep-tissue imaging because light diffusion limits the penetration depth [42]. Compared with optical imaging, MRI, CT, and PA imaging modalities are superior and have deeper penetration, but they show much lower spatial resolution [43,44]. To overcome these challenges, multimodality imaging techniques are proposed and have attracted increasing interest in recent years. Many studies have demonstrated that they possess the advantages of each imaging modality [43,45], therefore, they provide complementary information to improve the accuracy of disease diagnosis in vivo [43,46]. FL/MRI dual-modality AIEgen-based NPs can be prepared with decorations of gadolinium (III) and HIV-1 Tat peptide (RKKR-RORRRC) (Fig. 16 3D) [47]. TPETPAFN was incorporated into the matrices with amine and maleimide groups on the surface, which can be used for further surface modification. The amine groups were employed to react with diethylenetriaminepentaacetic dianhydride for chelation of Gd (III) as MR contrast. A cell-penetrating peptide, HIV-1 Tat, was covalently attached to the surface of AIE NPs through a maleimide thiol reaction. Fe3O4 core AIE/SiO2 shell (Fe3O4@AIE/ SiO2) hybrid NPs with fluorescent and magnetic features were prepared by hydrolysis and sol gel reaction of TEOS and silole-functionalized siloxane in the presence of citrate-coated Fe3O4 NPs (Fig. 16 3E) [48]. Benefiting from the advantages of AIEgens, the resultant Fe3O4@AIE/SiO2 NPs displayed strong magnetization and superior fluorescence.

16.3 Long-Term Cell Tracing With AIEgen-Based Fluorescent Nanoparticles A good material for long-term cell tracing should be easily delivered into cells with minimized leakage, and provide a detectable signal for a relatively long period of time without interfering the biological processes. AIEgen-based NPs are excellent candidates as cell tracers, since they usually have high brightness, superior photostability, excellent biocompatibility, and good cellular retention ability. Tat-TPETPAFN NPs prepared by nanoprecipitation method using AIE dye, TPETPAFN, as the fluorophore and lipid-PEG as matrix, are a typical example of AIEgen-based NPs. The cell-penetrating Tat peptide decoration on the surface of these NPs enables high cellular uptake efficiency. Tat-TPETPAFN NPs were highly emissive, with a QY of 24% in water compared to Qtracker655, a widely used QD-based commercial cell tracker which had a QY of 15%. The time-resolved scanning confocal fluorescence microscopy data suggested that, on average, the Tat TPETPAFN NPs were 10 times brighter than Qtracker 655 and did not have “blinking” problems, which are commonly observed in QDs. Tat-TPETPAFN NPs were also quite stable in the biological environment. When placed in cell culture medium, the NPs retained 93% of their initial fluorescence intensity after 9 days of incubation at 37 C. Qtracker 655, however, lost 58% of its initial intensity within 24 h. In the past few years, the application of AIEgen-based NPs for in vitro and in vivo long-term cell tracking were investigated extensively. Here we give a few representative examples.

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16.3.1 In Vitro Cell Tracing In the laboratories, cells are grown in a chemically-defined culture medium and a wellcontrolled environment. Tracking cultured cells in vitro allows us to study the dynamic cell behavior of various cell types in different but defined conditions. Also, the mobility or invasive and metastatic properties of tumor cells can be analyzed. More importantly, the study of cell cell interactions between chosen cell types is also possible. Cancer is a leading cause of death around the world. Long-term tracking of cancer cells helps researchers systematically and continuously monitor cancer cell migration, division, and lysis so as to study cancer pathogenesis and evaluate cancer treatment effects [5]. Spherical TPE-CS NPs with bright blue fluorescence and pH-dependent fluorescence were employed to label and trace HeLa cells (Fig. 16 4A) [33]. Experiments revealed that TPE-CS NPs can be internalized into cytoplasm through energy-dependent endocytosis and retained inside living cells for a long time. Only a small amount of the internalized NPs were excreted from the HeLa cells after 24 h of culture. TPE-CS NPs also showed good photostability with less than 25% signal loss upon 30 min of continuous excitation. The TPE-CS NPs can track the cells for seven passages. By employing AIEgen-based NPs with different emission colors, different populations of cells can be labeled with distinct colors. As shown in Fig. 16 4B, AIE NPs with BTPETD and TPETPAFN emitting green (539 nm) and far-red (670 nm) light, respectively, were used to label and trace two groups of HT1080 fibrosarcoma cells [24]. The cells labeled by these two types of AIE NPs were cultured together. In vitro cell imaging results indicated that the two groups of cells can be easily recognized by their specific emission colors (Fig. 16 4B). The internalized AIE NPs showed great stability and negligible leakage from the cells. The results indicated that it is possible to simultaneously track different cell populations and study cell-cell interactions using different AIE NPs with different emission colors. Stem cell-based therapy is regarded as the hope for some incurable diseases such as degenerative diseases, autoimmune diseases, and genetic disorders. Though promising, it is confronted with lots of challenges, due to limited understanding of the behavior and fate of stem cells. Inefficient and uncontrolled differentiation of stem cells could be disastrous. To achieve a better understanding of the mechanisms, therapeutic effect, and safety of stem cell-based therapy, tools and techniques for long-term tracking of stem cell migration and differentiation are highly desired. The application of AIEgen-based NPs for in vitro tracking of stem cell differentiation has been demonstrated [9]. Bone marrow-derived mesenchymal stem cells (BMSCs) can be effectively labeled and tracked by Tat peptide-decorated AIEgenbased NPs [9]. The AIE-Tat NPs were prepared by nanoprecipitation of a red-emitting TPE derivative (PITBT-TPE) together with DSPE PEG, followed by surface modification with Tat peptide. The use of AIE-Tat NPs made it possible for researchers to monitor the osteogenic differentiation without interference of cell viability and differentiation ability. Compared with Qtracker 655, a commercial cell tracker that can only track mouse BMSCs for six passages, AIE-Tat NPs were capable of tracking cells for over 12 passages. As a result, AIEgen-based NPs were demonstrated to be promising cell tracers for stem cell tracking.

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FIGURE 16–4 (A) Fabrication of TPE-CS NPs by ionic gelation method; relative FL intensity of CS NPs, TPE-CS, and TPE-CS NPs in an aqueous solution with different pH; HeLa cells stained with TPE-CS NPs [33]. (B) Dual-color cancer cell tracking by AIEgen-based NPs [24]. CS, chitosan; FL, fluorescence; NP, nanoparticle; TPE, tetraphenylethene. Reproduced from K. Li, Z. Zhu, P. Cai, R. Liu, N. Tomczak, D. Ding, et al., Organic dots with aggregation-induced emission (AIE dots) characteristics for dual-color cell tracing, Chem. Mater. 25 (21) (2013) 4181 4187; M. Li, Y. Hong, Z. Wang, S. Chen, M. Gao, R.T.K. Kwok, et al., Fabrication of chitosan nanoparticles with aggregationinduced emission characteristics and their applications in long-term live cell imaging, Macromol. Rapid Commun. 34 (9) (2013) 767 771, with permission. © 2013 American Chemical Society and 2013 Wiley, respectively.

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16.3.2 In Vivo Long-Term Cell Tracing In vitro cell imaging and tracking enable us to study cell behaviors under precisely controlled conditions. However, the actual in vivo environment is far more complicated. During longdistance cell migration, the cells may come across and/or respond to different physical barriers, chemical attractions, mechanical stresses, and cell cell communications. Noninvasive in vivo cell tracing provides more information and shows high biological significance. Most cancer deaths are caused by secondary metastatic tumor but not the initial tumor. Understanding how cancer cells escape, travel, and spread is critical for the study of both cancer etiology and cancer therapy. The in vivo noninvasive tracking of spreading tumor cells allows people to monitor the labeled cancer cells in real-time and obtain valuable information on cancer metastasis. To date, in vivo tracking of cancer cells has been achieved by various AIEgen-based NPs systems [14]. A typical example is the tracking of C6 glioma cancer cells in live mice with Tat-TPETPAFN NPs (Fig. 16 5) [14]. The C6 glioma cancer cells were first labeled with Tat-TPETPAFN NPs or Qtracker 655 as the positive control before being seeded into the mice. The FL signals from the AIE NPs or Qtracker 655 were monitored

FIGURE 16–5 Representative in vivo fluorescence images of C6 glioma cells by staining with (A) Tat-TPETPAFN NPs (Tat-AIE NPs) and (B) Qtracker 655 after injection for different days. (C) The integrated fluorescence intensities of the corresponding regions of interest (blue circles). The inset of HR-TEM shows the morphology of Tat-TPETPAFN NPs [14]. AIE, aggregation-induced emission; NP, nanoparticle; TPETPAFN, 2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl) phenyl)amino)phenyl)fumaronitrile. Reproduced from K. Li, W. Qin, D. Ding, N. Tomczak, J. Geng, R. Liu, et al., Photostable fluorescent organic dots with aggregation-induced emission (AIE dots) for noninvasive long-term cell tracing, Sci. Rep. 3 (2013) 1150, with permission. © 2013 Nature.

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during tumor growth. As shown in Fig. 16 5, the Tat-TPETPAFN NPs showed strong FL intensity and the FL signal remained detectable up to 21 days, while the Qtracker 655labeled cells became undetectable within 7 days. The Tat-TPETPAFN NPs had a large twophoton absorption cross-section value (6.7 3 105 GM) at 810 nm, so that two-photon imaging of tumor tissue with a deeper penetration depth was also possible. This work published in 2013 is the first demonstration of the AIEgen-based NPs for long-term in vivo cell tracing. The advantage of the AIEgen-based NPs over the commercial QD system was well presented. The cell type, therapeutic dose, and delivery route of stem cells used in the treatment may greatly affect the effectiveness of stem cell-based therapy. While harnessing the full therapeutic potential of stem cell-based therapy requires detailed study and clear elucidation of the in vivo stem cell behavior, noninvasive in vivo tracking and imaging of transplanted stem cells could provide insights into cell-based therapy for tissue regeneration. Stem cell tracking enables us not only to monitor, locate, and quantify transplanted cells, but also to trace cell migration and to follow the fate of transplanted stem cells in vivo. As introduced in the previous section, in vitro experiments demonstrated the great potential of AIEgen-based NPs for applications in long-term stem cell tracking [24]. The results encouraged the usage of these NPs in noninvasive in vivo long-term stem cell tracking [49]. Tat-TPETPAFN AIE NP-labeled adipose-derived stem cells (ADSCs) possessed stronger fluorescence and better retention ability compared to PKH26- and Qtracker 655-labeled stem cells, which are commercially available cell trackers. To evaluate their in vivo long-term cell tracking ability, the TatTPETPAFN NP-labeled ADSCs were intramuscularly injected in ischemic hindlimb-bearing mice and monitored in real-time during the healing process (Fig. 16 6). The results showed that the Tat-TPETPAFN AIE NPs were able to precisely track ADSCs for 42 days (Fig. 16 6C), which is the longest reported in vivo tracking duration achieved by an exogenous fluorescent cell tracer. The regeneration process can be monitored in real-time. To compare FP labeling and AIEgen-based NP labeling, GFP and Tat-TPETPAFN AIE NP duallabeled ADSCs were injected into ischemic hindlimb. The FL signals of the cells in the tissue were assessed 30 or 42 days after cell labeling and injection. It was observed that there was a good coincidence of Tat-TPETPAFN AIE NPs (red) and GFP (green) FL signals from the ADSCs in ischemic hindlimb slices. The signals from Tat-TPETPAFN AIE NPs were bright and gave good contrast for cell imaging and analysis.

16.4 Conclusions and Perspectives To date, various fluorescent materials have been developed and appliedin biological research and clinical studies. Highly emissive fluorescent materials that possess excellent photostability and biocompatibility hold great promise for bioapplications, such as bioimaging and cell tracing. This chapter introduced a group of newly emerged fluorescent NPs based on AIE fluorescent dyes which can be fabricated by the classical NP preparation methods. They are functionalized with specific molecules or chemical groups on their surfaces, and usually have outstanding optical properties and low cytotoxicity. In combination with fluorescence

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FIGURE 16–6 (A) Schematic structure of the Tat-TPETPAFN NPs; (B) in vitro ADSCs tracking by AIE dots, PKH26, or Qtracker 655; (C) in vivo tracking of AIE dot-labeled ADSC in mice; (D) in vivo tracking of ADSCs at single-cell resolution [49]. ADSC, adipose-derived stem cell; AIE, aggregation-induced emission; NP, nanoparticle; TPETPAFN, 2,3-bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)phenyl)fumaronitrile. Reproduced from D. Ding, D. Mao, K. Li, X. Wang, W. Qin, R. Liu, et al., Precise and long-term tracking of adipose-derived stem cells and their regenerative capacity via superb bright and stable organic nanodots, ACS Nano 8 (12) (2014) 12620 12631 with permission. © 2013 American Chemical Society.

microscopy, such as confocal microscopy, superresolution microscopy, and two-photon microscopy, samples can be imaged with high resolution and improved penetration depth. In this chapter, we focused on introducing the application of the AIEgen-based fluorescent NPs in the long-term imaging and tracing of cancer cells or stem cells. The field of AIEgen-based fluorescent NPs is promising but young. There still remains much room for further developing AIEgen-based fluorescent NPs with longer excitation/ emission wavelength, enhanced brightness and better stability. In the meantime, more detailed in vivo evaluations on the distribution, bio-stability, potential toxic effects, trafficking, retention, and clearance of AIEgen-based NPs in cells are expected. AIEgen-based NPs have enormous potential and a bright future. Here, we introduce the readers to the promising fluorescent nanomaterials, and we hope that this chapter will give inspiration to material scientists and biologists.

References [1] S. Chen, H. Wang, Y. Hong, B.Z. Tang, Fabrication of fluorescent nanoparticles based on AIE luminogens (AIE dots) and their applications in bioimaging, Mater. Horiz. 3 (4) (2016) 283 293. [2] X. Trepat, Z. Chen, K. Jacobson, Cell migration, Compr. Physiol. 2 (4) (2012) 2369 2392.

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[3] C.L. Chaffer, R.A. Weinberg, A perspective on cancer cell metastasis, Science 331 (6024) (2011) 1559 1564. [4] M.F. Kircher, S.S. Gambhir, J. Grimm, Noninvasive cell-tracking methods, Nat. Rev. Clin. Oncol. 8 (11) (2011) 677 688. [5] Z. Wang, S. Chen, J.W.Y. Lam, W. Qin, R.T.K. Kwok, N. Xie, et al., Long-term fluorescent cellular tracing by the aggregates of AIE bioconjugates, J. Am. Chem. Soc. 135 (22) (2013) 8238 8245. [6] E. Li, X. Cheng, Y. Deng, J. Zhu, X. Xu, P.E. Saw, et al., Fabrication of PEGylated Fe@Bi2S3 nanocomposites for dual-mode imaging and synergistic thermoradiotherapy, Biomater. Sci. 6 (7) (2018) 1892 1898. [7] X. Liu, X. Zhang, M. Zhu, G. Lin, J. Liu, Z. Zhou, et al., PEGylated Au@Pt nanodendrites as novel theranostic agents for computed tomography imaging and photothermal/radiation synergistic therapy, ACS Appl. Mater. Interfaces 9 (1) (2017) 279 285. [8] X. Tian, L. Zhang, M. Yang, L. Bai, Y. Dai, Z. Yu, et al., Functional magnetic hybrid nanomaterials for biomedical diagnosis and treatment, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 10 (1) (2018) e1476. [9] M. Gao, J. Chen, G. Lin, S. Li, L. Wang, A. Qin, et al., Long-term tracking of the osteogenic differentiation of mouse BMSCs by aggregation-induced emission nanoparticles, ACS Appl. Mater. Interfaces 8 (28) (2016) 17878 17884. [10] Y. Yu, C. Feng, Y. Hong, J. Liu, S. Chen, K.M. Ng, et al., Cytophilic fluorescent bioprobes for long-term cell tracking, Adv. Mater. 23 (29) (2011) 3298 3302. [11] G. Feng, C.Y. Tay, Q.X. Chui, R. Liu, N. Tomczak, J. Liu, et al., Ultrabright organic dots with aggregationinduced emission characteristics for cell tracking, Biomaterials 35 (30) (2014) 8669 8677. [12] Z. Zhao, S. Chen, J.W.Y. Lam, C.K.W. Jim, C.Y.K. Chan, Z. Wang, et al., Steric hindrance, electronic communication, and energy transfer in the photo- and electroluminescence processes of aggregationinduced emission luminogens, J. Phys. Chem. C 114 (17) (2010) 7963 7972. [13] Z. Zhao, C. Deng, S. Chen, J.W.Y. Lam, W. Qin, P. Lu, et al., Full emission color tuning in luminogens constructed from tetraphenylethene, benzo-2,1,3-thiadiazole and thiophene building blocks, Chem. Commun. 47 (31) (2011) 8847 8849. [14] K. Li, W. Qin, D. Ding, N. Tomczak, J. Geng, R. Liu, et al., Photostable fluorescent organic dots with aggregation-induced emission (AIE dots) for noninvasive long-term cell tracing, Sci. Rep. 3 (2013) 1150. [15] N. Zhao, Z. Yang, J.W.Y. Lam, H.H.Y. Sung, N. Xie, S. Chen, et al., Benzothiazolium-functionalized tetraphenylethene: an AIE luminogen with tunable solid-state emission, Chem. Commun. 48 (69) (2012) 8637 8639. [16] H. Gao, X. Zhao, S. Chen, AIEgen-based fluorescent nanomaterials: fabrication and biological applications, Molecules 23 (2) (2018) 419. [17] S.M.A. Fateminia, Z. Wang, C.C. Goh, P.N. Manghnani, W. Wu, D. Mao, et al., Nanocrystallization: a unique approach to yield bright organic nanocrystals for biological applications, Adv. Mater. 29 (1) (2017) 1604100. [18] Q. Zhao, K. Li, S. Chen, A. Qin, D. Ding, S. Zhang, et al., Aggregation-induced red-NIR emission organic nanoparticles as effective and photostable fluorescent probes for bioimaging, J. Mater. Chem. 22 (30) (2012) 15128 15135. [19] W. Qin, D. Ding, J. Liu, W.Z. Yuan, Y. Hu, B. Liu, et al., Biocompatible nanoparticles with aggregationinduced emission characteristics as far-red/near-infrared fluorescent bioprobes for in vitro and in vivo imaging applications, Adv. Funct. Mater. 22 (4) (2012) 771 779. [20] X. Wang, P. Song, L. Peng, A. Tong, Y. Xiang, Aggregation-induced emission luminogen-embedded silica nanoparticles containing DNA aptamers for targeted cell imaging, ACS Appl. Mater. Interfaces 8 (1) (2016) 609 616. [21] F. Zhang, T. Zhou, G. Liu, J. Shi, H.Z. Zhong, Y. Dong, Tetraphenylethylene derivative capped CH3NH3PbBr3 nanocrystals: AIE-activated assembly into superstructures, Faraday Discuss. 196 (2017) 91 99.

374

THERANOSTIC BIONANOMATERIALS

[22] Q. Wan, M. Liu, D. Xu, L. Mao, J. Tian, H. Huang, et al., Fabrication of aggregation induced emission active luminescent chitosan nanoparticles via a “one-pot” multicomponent reaction, Carbohydr. Polym. 152 (2016) 189 195. [23] X. Fang, X. Chen, R. Li, Z. Liu, H. Chen, Z. Sun, et al., Multicolor photo-crosslinkable AIEgens toward compact nanodots for subcellular imaging and STED nanoscopy, Small 13 (41) (2017) 1702128. [24] K. Li, Z. Zhu, P. Cai, R. Liu, N. Tomczak, D. Ding, et al., Organic dots with aggregation-induced emission (AIE dots) characteristics for dual-color cell tracing, Chem. Mater. 25 (21) (2013) 4181 4187. [25] D. Wang, J. Qian, W. Qin, A. Qin, B.Z. Tang, S. He, Biocompatible and photostable AIE dots with red emission for in vivo two-photon bioimaging, Sci. Rep. 4 (2014) 4279. [26] J. Geng, K. Li, D. Ding, X. Zhang, W. Qin, J. Liu, et al., Lipid-PEG-folate encapsulated nanoparticles with aggregation induced emission characteristics: cellular uptake mechanism and two-photon fluorescence imaging, Small 8 (23) (2012) 3655 3663. [27] R. Yadav, D. Kumar, A. Kumari, S.K. Yadav, Encapsulation of catechin and epicatechin on BSA NPS improved their stability and antioxidant potential, Excli. J. 13 (2014) 331 346. [28] L. Liu, F. Zhang, B. Xu, W. Tian, Silica nanoparticles based on an AIE-active molecule for ratiometric detection of RNS in vitro, J. Mater. Chem. B 5 (46) (2017) 9197 9203. [29] D. Li, W. Qin, B. Xu, J. Qian, B.Z. Tang, AIE nanoparticles with high stimulated emission depletion efficiency and photobleaching resistance for long-term super-resolution bioimaging, Adv. Mater. 29 (43) (2017) 1703643. [30] Z. Chen, J. Zhang, M. Song, J. Yin, G. Yu, S.H. Liu, A novel fluorene-based aggregation-induced emission (AIE)-active gold(i) complex with crystallization-induced emission enhancement (CIEE) and reversible mechanochromism characteristics, Chem. Commun. 51 (2) (2015) 326 329. [31] B. Yang, X. Zhang, X. Zhang, Z. Huang, Y. Wei, L. Tao, Fabrication of aggregation-induced emission based fluorescent nanoparticles and their biological imaging application: recent progress and perspectives, Mater. Today 19 (5) (2016) 284 291. [32] C. Ma, G. Xie, X. Zhang, L. Yang, Y. Li, H. Liu, et al., Ring-opening crosslinking PEGylation of an AIE epoxy monomer towards biocompatible fluorescent nanoparticles, J. Mater. Chem. B 4 (48) (2016) 8009 8015. [33] M. Li, Y. Hong, Z. Wang, S. Chen, M. Gao, R.T.K. Kwok, et al., Fabrication of chitosan nanoparticles with aggregation-induced emission characteristics and their applications in long-term live cell imaging, Macromol. Rapid Commun. 34 (9) (2013) 767 771. [34] M. Faisal, Y. Hong, J. Liu, Y. Yu, J.W. Lam, A. Qin, et al., Fabrication of fluorescent silica nanoparticles hybridized with AIE luminogens and exploration of their applications as nanobiosensors in intracellular imaging, Chem. Eur. J. 16 (14) (2010) 4266 4272. [35] G. Feng, B. Liu, Aggregation-induced emission (AIE) dots: emerging theranostic nanolights, Acc. Chem. Res. 51 (6) (2018) 1404 1414. [36] P.S. Low, W.A. Henne, D.D. Doorneweerd, Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases, Acc. Chem. Res. 41 (1) (2008) 120 129. [37] J. Cheon, J. Lee, Synergistically integrated nanoparticles as multimodal probes for nanobiotechnology, Acc. Chem. Res. 41 (12) (2008) 1630 1640. [38] J. Qian, B.Z. Tang, AIE luminogens for bioimaging and theranostics: from organelles to animals, Chem 3 (1) (2017) 56 91. [39] M. Li, J.W.Y. Lam, F. Mahtab, S. Chen, W. Zhang, Y. Hong, et al., Biotin-decorated fluorescent silica nanoparticles with aggregation-induced emission characteristics: fabrication, cytotoxicity and biological applications, J. Mater. Chem. B 1 (5) (2013) 676 684. [40] A. Louie, Multimodality imaging probes: design and challenges, Chem. Rev. 110 (5) (2010) 3146 3195.

Chapter 16 • AIEgen-Based Fluorescent Nanoparticles for Long-Term Cell Tracing

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[41] J. Kim, Y. Piao, T. Hyeon, Multifunctional nanostructured materials for multimodal imaging, and simultaneous imaging and therapy, Chem. Soc. Rev. 38 (2009) 372 390. [42] J. Zhang, C. Li, X. Zhang, S. Huo, S. Jin, F. An, et al., In vivo tumor-targeted dual-modal fluorescence/ CT imaging using a nanoprobe co-loaded with an aggregation-induced emission dye and gold nanoparticles, Biomaterials 42 (2015) 103 111. [43] X. Yi, J. Li, Z. Zhu, Q. Liu, Q. Xue, D. Ding, In vivo cancer research using aggregation-induced emission organic nanoparticles, Drug Discov. Today 22 (9) (2017) 1412 1420. [44] J. Geng, L. Liao, W. Qin, B.Z. Tang, N. Thakor, B. Liu, Fluorogens with aggregation induced emission: ideal photoacoustic contrast reagents due to intramolecular rotation, J. Nanosci. Nanotechnol. 15 (2) (2015) 1864 1868. [45] T. Nam, S. Park, S. Lee, K. Park, K. Choi, I.C. Song, et al., Tumor targeting chitosan nanoparticles for dual-modality optical/MR cancer imaging, Bioconjug. Chem. 21 (4) (2010) 578 582. [46] H. Xing, W. Bu, S. Zhang, X. Zheng, M. Li, F. Chen, et al., Multifunctional nanoprobes for upconversion fluorescence, MR and CT trimodal imaging, Biomaterials 33 (4) (2012) 1079 1089. [47] K. Li, D. Ding, C. Prashant, W. Qin, C. Yang, B.Z. Tang, et al., Gadolinium-functionalized aggregationinduced emission dots as dual-modality probes for cancer metastasis study, Adv. Healthc. Mater. 2 (12) (2013) 1600 1605. [48] F. Mahtab, Y. Yu, J.W.Y. Lam, J. Liu, B. Zhang, P. Lu, et al., Fabrication of silica nanoparticles with both efficient fluorescence and strong magnetization, and exploration of their biological applications, Adv. Funct. Mater. 21 (9) (2011) 1733 1740. [49] D. Ding, D. Mao, K. Li, X. Wang, W. Qin, R. Liu, et al., Precise and long-term tracking of adiposederived stem cells and their regenerative capacity via superb bright and stable organic nanodots, ACS Nano 8 (12) (2014) 12620 12631.

17 Multimodal Carbon Dots as Biosensors Jisu Hong
, Mirae Kim
, Chaenyung Cha SCHOOL OF MATERIALS SCIENCE AND ENGINEERING, ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY, ULSAN, SOUT H K OREA

17.1 Introduction Recent advances in nanotechnology, which has garnered worldwide attention not just from the scientific community but also from the general public, have been largely due to the promise that new functionalities could be attained from existing materials in nanoscale dimensions. One such area is optics, in which various optical phenomena such as surface plasmonics, photoluminescence (PL), photon upconversion, and surface-enhanced Raman scattering (SERS) could be observed and even manipulated at the nanoscale [13]. For example, graphitic nanomaterials [e.g., graphene, carbon nanotubes (CNTs)] due to their extensive sp2-carbon system give rise to near-infrared (NIR) fluorescence and SERS [4]. Semiconductorbased nanocrystals (“quantum dots”), such as CdSe and ZnS, demonstrate highly stable and energy-efficient fluorescence by tuning their size and chemical compositions [5]. In 2004, Scrivens et al. introduced a new class of carbon-based nanomaterial, a heterogeneous mixture of residual carbon nanostructures isolated from a reaction mixture for CNTs demonstrating fluorescence under ultraviolet (UV) excitation [6]. Ever since, numerous research efforts have been exerted to develop these fluorescent carbon-based nanostructures from various carbon sources and tunable fluorescence emission. Commonly and collectively referred to as carbon dots (CDs) to highlight the similar PL properties and also the difference in chemical makeup compared to semiconductor quantum dots (SQDs), CDs have piqued the interest of many scientists and engineers due to their unique blend of characteristics that could potentially be advantageous over SQDs [79]. Unlike SQDs, CDs can be prepared from various inexpensive carbon sources, from graphitic carbons to small molecules and polymers. Their highly efficient mass production is also possible due to simple fabrication techniques. CDs are considered especially promising in the field of biomedical engineering, especially in bioimaging and biosensing, mainly for their biocompatibility compared to SQDs whose potential biomedical applications have been largely quelled, despite their popularity, due to the concern over potential cytotoxicity arising from the heavy metal-based source material [10].


These authors contributed equally.

Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00017-1 © 2019 Elsevier Inc. All rights reserved.

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In addition, CDs can be further modified to impart multifunctionality through numerous functional groups on their surface; targeting ligands, drug molecules, imaging modalities for multimodal imaging, and theranostic effects. In this chapter, the synthesis and various physicochemical properties of CDs, especially in relation to their unique optical phenomena, are discussed in detail. In addition, several examples of recent research efforts into CD technology and its biomedical applications are also introduced.

17.2 Classification of Carbon Dots and Origins of Photoluminescence 17.2.1 Classification and Nomenclature of Carbon Dots With the number of scientific publications in CD research exploding only within the last decade, as well as the very heterogeneous nature of the CDs themselves, the field of CD research has not yet come to a consensus regarding the scientific classification of CDs. This is understandable considering the relative infancy of the field, but nonetheless it is important to accurately characterize the physicochemical properties of CDs prepared from a diverse array of sources and methods, and more firmly and coherently classify the CDs based on their common features for systematic investigation [11,12]. Also, the classification helps decrease the confusion around the usage of terminology, generate open and insightful discussion, and eventually legitimize CD as a bona fide research field going forward. CDs have been commonly named simply based on their carbon source: graphene quantum dot (GQD), carbon quantum dot (CQD) and carbon nanodot (CND) derived from graphitic carbons (e.g., graphene sheets, CNTs, and fullerenes), and polymer dots (PDs) derived from small molecules and polymers (Table 171, Fig. 171) [11,12]. GQDs, CQDs, and CNDs are classified based on their chemical structure; planar sp2-carbon (GQDs and CQDs) versus a heterogeneous mixture of sp3- and sp2-carbon (CNDs). GQDs are composed of a few graphene layers connected through chemical bonds formed at the edges, with overall anisotropic shape (i.e., lateral dimensions along the graphene layers are larger than their stacked height). CQDs are more spherical, with a greater number of sp2-carbon layers, and often contain more sp3-carbons than GQDs. Unlike GQDs and CQGs, CNDs are more spherical and mostly made of sp3-carbons (and thus lack crystallinity). In addition, CNDs and CQDs can be obtained from both graphitic carbons and organic molecules and polymers, whereas GQDs are obtained only from graphitic carbons. PDs are similar to CNDs in terms of the carbon structure, having dominant noncrystalline sp3-carbons, but PDs retain more native crosslinked polymeric network structures that are often aggregated to form nanostructures. In addition, PDs are fabricated from a wide range of polymers and small organic molecules, leading to structural heterogeneity, whereas CNDs can be also derived from graphitic carbons.

Chapter 17 • Multimodal Carbon Dots as Biosensors

Table 17–1

Characteristics of Different Types of Carbon Dots SQDs

GQDs

Carbon source

N/A (inorganic)

Graphitic carbon

Shape Light absorption

Sphere (hard) UV and visible range (broad)

Light emission

PL mechanism

379

CQDs

CNDs

Graphitic carbon Polymer Polymer Small Small molecule molecule Sphere (soft)

Disc UV range (broad) UV range (broad) Can red shift Major peak B upon passivation 230 nm (π!π
) Down-conversion PL Down-conversion PL Down-conversion PL Phosphorescence Upconversion PL Upconversion PL Phosphorescence Quantum Quantum confinement Collective confinement Collective exciton exciton

Size-dependent PL Yes Excitation-dependent PL No

PDs

Yes

Collective exciton Individual emitter

No Yes

CQDs, carbon quantum dots; CNDs, carbon nanodots; GQDs, graphene quantum dots; PL, photoluminescence; PDs, polymer dots; SQDs, semiconductor quantum dots.

FIGURE 17–1 Classification of CDs: GQDs, CQDs, and CNDs. CDs, Carbon dots; CQDs, carbon quantum dots; CNDs, carbon nanodots; GQDs, graphene quantum dots.

17.2.2 Origins of Photoluminescence of Carbon Dots 17.2.2.1 Quantum Confinement Effect and Collective Exciton Effect Regardless of the synthetic routes and source materials, the term “quantum” is added to the types of CDs, such as GQDs and CQDs, that contain crystalline sp2-carbon structures and therefore demonstrate PL from a quantum confinement effect, as opposed to CNDs whose PL is based upon a different mechanism. The quantum confinement effect occurs when the size of a material is smaller than the Bohr exciton radius and within the same magnitude as the de Broglie wavelength of the electron, in which there is a discrete size-dependent energy bandgap between valence and conduction bands [11,12]. Therefore, upon absorption of a photon resulting in an electron from HOMO of the valence band being promoted to LUMO of the conduction band, there is emission of the photon, fluorescence, which blueshifts with

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decreasing particle size. For SQDs with well-defined crystalline structure, the bandgap is discrete and the size distribution is much narrower than CDs. In this case, the PL is dominantly governed by the quantum confinement effect, with narrow emission band and excitationindependent emission (i.e., emission has a maximum regardless of the excitation wavelength) (Fig. 172A). For CQDs and GQDs, there are other energy states, called “trap states,” within the bandgap that the electrons can occupy, owing to their heterogeneity (e.g., size variation, surface defects, doping, functional groups, presence of both sp2 and sp3 carbons, etc.). In this case, the PL occurs via the “collective exciton effect” in which exciton and hole can be trapped in these states and their recombination results in emission at lower energy states, in addition to the quantum confinement effect (Fig. 172B). The collective exciton effect usually occurs at the surface where the defects and other functional groups are mostly present, whereas the quantum confinement effect mostly occurs in the core region which is made of mostly sp2carbon structures. Both CQDs and GQDs show wide emission spectra, from visible to near IR range, and excitation-dependent emission (i.e., tunable emission under excitation at different wavelengths). For CNDs which lack a sp2-carbon core that CQDs possess, the PL is not governed by the quantum confinement effect nor the collective exciton effect. Rather, individual fluorophores and/or surface functional groups give rise to several energy states that excitons can occupy (i.e., emitters), and the superposition of different emissions from these energy states lead to a broad emission spectrum (Fig. 172C). In addition, due to the lack of a well-defined bandgap and combined exciton effect, the emission spectrum is not strongly dependent on the particle size. It should be noted that CNDs that do possess a lesser extent of sp2-carbon core than CQDs have been shown to behave similar to excitation-dependent emission. PDs,

FIGURE 17–2 PL mechanisms of CDs: (A) quantum confinement effect, (B) collective exciton effect, (C) individual emitters. (D) Two different edge shapes of graphene: armchair and zigzag. CDs, carbon dots.

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which contain a carbonized core with crosslinked polymeric surface with numerous functional groups, also display PL by the same mechanism. Interestingly, the PL behavior of PDs is further governed by a crosslinking-enhanced effect, in which the crosslinked polymeric network decreases the extent of excitons returning to the ground state by nonradiative vibration and rotation, thereby increasing the radiative PL [13]. Collectively, it should be emphasized that the PL behavior of CDs is highly dependent on the mode of synthesis, carbon source, and postmodifications, which all significantly dictate their heterogeneity, and thus these factors are actively explored to control their properties.

17.2.2.2 Surface/Edge State of Graphene Quantum Dots A diverse array of edge shapes of GQDs is obtained after synthesis, such as zigzags and armchair edges. The chemical and physical structures of these edges play a significant role in the PL properties of GQDs by altering their energy states. It has been reported that the zigzag edges are carbenes with a triplet ground state, whereas armchair edges are carbynes with a singlet ground state (Fig. 172D) [14]. As such, GQDs with a greater portion of zigzag edges display smaller energy bandgap than those with armchair edges. Pan et al. developed ultrafine, single-layer GQDs via hydrothermal treatment of graphene oxide (GO) sheets, with dominant triplet zigzag edges [15]. Due to the ground-state multiplicity of singlet and triplet carbine, two electronic transitions were observed at 257 and 320 nm, and blue PL with peak emission intensity at 430 nm which corresponded to the transition was demonstrated. Interestingly, at acidic pH the protonation of carbine resulted in the breakup of emissive triplet energy state leading to PL quenching, which was reversible upon increasing pH. Lin and Zhang similarly demonstrated the synthesis of GQDs having two different sizes (9.6 and 20 nm) with similar zigzag-edge driven PL behavior, which signified the dominant effect of the zigzag edge state over the quantum confinement effect [16].

17.3 Synthesis of Carbon Dots The synthetic approaches can be classified into “top-down” and “bottom-up” (Fig. 173). Top-down approaches refer to the scaling down of a bulk carbon source to nanometer-scale carbon particles, whereas bottom-up approaches involve the assembly of dispersed carbon precursors (e.g., small molecules, polymers, and CNTs), usually in a solvent, into nanostructures using such methods as high energy treatment, templating, and self-assembly. Regardless, the underlying theme for the synthesis of CDs, like any other carbon nanomaterials that have been extensively investigated in the last three decades, is the involvement of high-energy treatment to a particular carbon source for carbonization.

17.3.1 “Top-Down” Approaches 17.3.1.1 Laser Ablation Laser ablation refers to the process of removing a certain portion of a material from the surface by high-intensity laser treatment. It is widely used in many industrial settings, such as

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FIGURE 17–3 Various methods to synthesize CDs: (A) “top-down” and (B) “bottom-up” approaches. CDs, carbon dots.

laser machining and drilling, and special coatings (e.g., pulsed laser deposition), as it allows a controllable amount of energy in duration and dosage to be applied to the specific target area. Laser ablation is no stranger to carbon research, as it is commonly utilized to synthesize various carbon nanomaterials, such as CNTs and fullerenes [17]. Kroto, Curl, and Smalley famously synthesized spherical fullerenes (C60) by vaporizing graphite via pulsed laser irradiation (second harmonic of Q-switched Nd:YAG laser), which has since became a Nobel prize winning work [18]. Smalley et al. also first reported the synthesis of CNTs from a graphitic block containing catalytic metals (e.g., Co, Pt) by applying laser at high temperature (1200 C) [19]. Mainly due to the widespread usage in carbon research, laser ablation has been extensively adopted to prepare CDs. Sun et al. were one of the first to apply laser ablation to carbon source (baked graphite with cement) to produce CDs [20]. The laser ablation itself only produced a heterogeneous mixture of carbon nanoparticles without PL, suggesting the carbonized core was mostly made of sp3-carbon. The nanoparticles only became CDs after surface passivation with poly(ethylene glycol) and poly(propionylethyleneimine-coethyleneimine). More scalable and faithful production of photoluminescent CDs directly from laser ablation has been demonstrated by using carbon suspensions in different solvents, either by themselves or those containing organic molecules or polymers, that can directly passivate the carbon nanoparticles, rather than solid carbon materials. For example, Li et al. demonstrated that graphite flakes dispersed in ethanol and acetone could be converted to CQDs via pulsed laser ablation, with spherical crystalline multishell fullerene morphology [21]. Similarly, Hu et al. created CQDs from pulsed laser irradiation of graphite flakes in poly(ethylene glycol) solution [22].

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17.3.1.2 Arc Discharge In a typical arc discharge setting, electrical current is passed between graphitic electrodes with 1 mm or less spacing under inert argon atmosphere [23]. The carbon nanoparticles generated from the vaporization of the carbon electrode are deposited on the cathode. Like laser ablation, the technique itself is fairly simple and straightforward, but the product is a highly heterogeneous mixture of carbon nanoparticles, and thus often poses the challenge of purifying CDs from the mixture with low yield. The first known report of CDs by Scrivens et al. isolated photoluminescent CDs from carbon nanoparticle mixture originally intended to synthesize CNTs [6]. The CDs fabricated from arc discharge of CNTs were identified to be a mixture of GQDs and CQDs, depending on the oxidation and the molecular weight of the CNTs.

17.3.1.3 Electrochemical Carbonization Similar to arc discharge, electrical potential could be applied between a carbon anode and a cathode, but in an electrolyte solution, resulting in exfoliation of graphitic carbon source to form, as demonstrated by Lu et al. [24]. The mechanism of GQD generation is known to be oxidative cleavage of carbon anode coupled with intercalation of ions. Tan et al. similarly synthesized red-fluorescent GQDs with a narrow size distribution from electrochemical exfoliation of graphite dispersed in potassium persulfate solution [25]. The radicals generated from persulfate were responsible for “cutting” graphene sheets into smaller, intact sp2-carbon structures.

17.3.2 “Bottom-Up” Approaches 17.3.2.1 Pyrolysis—Solvothermal and Hydrothermal Carbonization Pyrolysis is a highly effective and efficient method of breaking down a larger graphitic carbon to smaller, reactive fragments which would eventually be used to build various carbon nanomaterials, including CNTs, fullerenes, and nanodiamonds [26]. Conversely, pyrolysis can be used to develop carbon nanomaterials from smaller carbon sources such as organic molecules and polymers because the high energy generated from the pyrolysis leads to the activation of reactive species capable of fusion with other molecules during carbonization. For this reason, CDs are widely prepared by pyrolysis from various carbon sources. The process is either termed solvothermal carbonization (STC) or hydrothermal carbonization (HTC), depending on the type of solvent used to disperse the carbon precursors: STC for organic solvents and HTC for water. Pyrolysis alone is often not sufficient to produce scalable CDs as it usually results in a heterogeneous mixture containing non-PL particles. Therefore, the pyrolysis is done under acidic or basic conditions at high temperature to break down carbon precursors and assemble CDs with simultaneous passivation. For example, Wang et al. demonstrated that CQDs with high quantum yield and tunable emission could be developed from STC (300 C for 3 h) of citric acid as a carbon source in octadecene as a solvent and hexadecylamine as a surface passivating agent [27]. Similarly, Zhu et al. reported the synthesis of GQDs by treating GO dispersion in dimethylformamide at 300 C for 3 h [28].

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One critical advantage of pyrolysis over other synthetic methods is the ability to use a variety of carbon precursors. While top-down approaches generally use graphitic carbon materials as precursors, pyrolysis can be applied to virtually any carbon-containing small organic molecules, oligomers, and polymers that can be dispersed in suitable solvents. The carbon precursors include but are not limited to natural polymers (e.g., chitosan, alginate, proteins) and organic molecules (e.g., ascorbic acid, citric acid, glucose) that are mostly aqueous-soluble, and thus converted to CDs via HTC [2931]. For example, Yang et al. synthesized CQDs via HTC of glucose solution in the presence of phosphate ions, which allowed the control of emission spectrum by controlling their size [31]. Interestingly, rather than purified molecules, several research groups have demonstrated that inexpensive, unprocessed raw materials (e.g., cellulosic biomass) could be converted to CDs via pyrolysis, demonstrating an environmentally friendly synthetic route [32].

17.3.2.2 Microwave/Ultrasonic-Assisted Method More recently, microwave-assisted HTC has been actively adopted to generate CDs in a rapid and cost-effective manner, since microwave allows the heating to occur at shorter time periods (in a matter of minutes), even with a commercial microwave oven, opening up the possibility of large-scale industrial production of CDs. Zhu et al. were one of the first to demonstrate the use of microwave (500 W, 210 min) to synthesize CNDs with tunable emission spectrum from carbohydrate solution (e.g., glucose and fructose) containing varying amounts of poly(ethylene glycol) as a passivating agent [33]. Similarly, Zhai et al. also demonstrated the synthesis of CNDs, by applying microwave (700 W, up to 2 min) irradiation to citric acid solution (1%, 10 mL) containing ethylenediamine as a surface passivating agent [34]. Similarly, ultrasonic treatment can be used to carry out HTC to develop CDs, as it can serve the same purpose of applying high energy in short durations that microwave treatment can provide. Li et al. demonstrated that CNPs could be synthesized by applying ultrasonication to an activated carbon dispersed in hydrogen peroxide solution [35].

17.3.2.3 Template-Supported Method Generally, CDs are prepared in situ from carbon sources in solution or atmospheric conditions, thereby presenting inherent size distribution regardless of the synthetic approach. Several researchers have introduced the strategy of using porous supports such as mesoporous silica, alumina, and metal-organic framework (MOF) as templates upon which CDs are synthesized. Zong et al. were able to produce CQDs having a narrow size distribution (1.52.5 nm) via HTC of citric acid performed within the nanoscale pores (average diameter of 3.6 nm) of silica nanospheres [36]. The silica nanosphere templates could be easily removed by etching in basic solution to obtain high-purity CQDs. More recently, Bhattacharyya et al. were able to synthesize N-doped CDs directly by carbonizing anodic MOF containing dimethylamine cations via pyrolysis at 500 C [37]. Similar to silica nanospheres, residual MOF could be easily removed by acidic solution.

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17.4 Spectroscopic Properties of Carbon Dots 17.4.1 UV Absorption CDs typically show a very strong absorbance at UV region. The exact spectral position depends on various factors: size, type and extent of carbon bonds (sp2 vs sp3), solvent, and passivation. Generally, for those with more ordered sp2-carbon, such as GQD and CQD, the maximum absorption usually peaks around 250300 nm via π!π
transition, coinciding with the absorption of aromatic π system. However, the spectrum can vary, with the maximum occurring at longer wavelengths, depending largely on the surface passivation. The presence of other functional groups could lead to π-electron delocalization of the sp2 carbon system. For example, carbonyl (CQO) or carbonnitrogen (CN) bonds having electrons at a higher energy level (“n state”) than the π state has been shown to result in absorption at even higher wavelengths via n!π
transition, from 350 nm to visible ranges (Fig. 174A) [34]. Also, other heteroatom doping, most notably B- and S-doping, of carbon nanomaterials can significantly alter their electronic properties. For more amorphous CNDs and PDs, the absorption spectrum can vary more widely depending on reaction conditions.

17.4.2 Photoluminescence The very essence of CDs lies with the ability to emit light at a visible range after absorbing light at UV range. Therefore, the primary objective for CD research is to reliably tune their PL properties by controlling various synthetic parameters.

FIGURE 17–4 (A) Absorption behavior of N-doped CDs. Two distinct absorption peaks corresponding to π!π
and n!π
transitions were observed. (B) Excitation-dependent emission spectra of CDs. CDs, carbon dots. (A) Reproduced from X. Zhai, P. Zhang, C. Liu, T. Bai, W. Li, L. Dai, et al., Highly luminescent carbon nanodots by microwave-assisted pyrolysis, Chem. Commun. 48 (2012) 79557957 with permission from The Royal Society of Chemistry, © 2017. (B) Reproduced from F. Wang, S. Pang, L. Wang, Q. Li, M. Kreiter, C.-y. Liu, One-step synthesis of highly luminescent carbon dots in noncoordinating solvents, Chem. Mater. 22 (2010) 45284530 with permission from the American Chemical Society, © 2010.

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One of the most unique aspects of PL of CDs, compared to other known fluorophores, is the excitation-dependence of emission intensity and wavelength (Fig. 174B). This is primarily due to the quantum confinement effect caused by the size of CDs, and various trap states causing recombination of excitons (“collective exciton effect”). As a result, there is a common photoluminescent feature for all CDs; the increasing emission wavelength and decreasing emission intensity with absorption wavelength. The size and shape of CDs have been shown to influence photoluminescent behavior, especially for GQDs, as the size-dependent quantum confinement effects are more magnified in sp2-carbon-heavy GQDs. With increasing size in the nanoscale, the energy bandgap generally decreases, and therefore the emission occurs at longer wavelengths (“red-shift”). Kim et al. demonstrated this size-dependent effect of GQDs by varying their average size (Fig. 175) [38]. Here, they showed that with the excitation at 325 nm, PL emission redshifted with increasing GQD size from 5 to 35 nm. In addition, within the same size range, the maximum absorption occurred at longer wavelengths and the intensity decreased with increasing GQD size. The shape of the GQDs also dictated their edge states, showing increased armchair edges when the size increased above 17 nm, during which the overall shape was changed from circular to polygonal. As a result, the emission intensity increased with size above 17 nm.

17.4.3 Upconversion Photoluminescence Tunable PL is a hallmark of CDs. More recent research progress has demonstrated that this tunability could be extended beyond the visible range. "Upconversion" of photons occurs when simultaneous absorption of multiple photons leads to the emission of light at a shorter wavelength than the excitation wavelength. There is a select group of molecules and ions that can induce upconversion, such as ions of d-block and f-block elements (e.g., Ln31, Mo31, and Re41). However, several research groups have demonstrated the upconversion PL (UCPL) from CQDs and GQDs. Zhuo et al. developed GQDs from ultrasonic-assisted hydrothermal pyrolysis of GO in acidic solution showed excitation-dependent UCPL, in addition to typical UV-excited PL, in which upconverted emission at 407 nm with the excitation was at visible range from 500 to 700 nm (Fig. 176A) [39]. Deng et al. similarly demonstrated CQDs prepared from hydrothermal pyrolysis of ascorbic acid as a carbon source and Cu21, which showed UCPL from the excitation at near-IR range (8001000 nm) [40].

17.4.4 Phosphorescence Many of the PL properties of CDs have been focused on fluorescence, emission from singletsinglet relaxation. But recent studies suggest some of the CDs also display phosphorescent PL, emission from triplet-singlet relaxation. Deng et al. first reported the phosphorescence of CQDs from pyrolysis of ethylenediaminetetraacetic acid [41]. The phosphorescence with a long lifetime (380 ms) could be achieved by embedding CQDs in a poly(vinyl alcohol) matrix which helps stabilize the triplet state and prevent nonradiative relaxation via hydrogen bonding. The mechanism of phosphorescence of CDs is thought to originate from multiple

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FIGURE 17–5 Size- and shape-dependent PL behavior of GQDs. With increasing size, the emission spectra of GQDs red-shifted from 5 to 35 nm. Above 17 nm, the overall shape became polygonal with larger armchair edges, resulting in increased emission intensity. GQDs, graphene quantum dots; PL, photoluminescence. Reproduced from S. Kim, S.W. Hwang, M.-K. Kim, D.Y. Shin, D.H. Shin, C.O. Kim, et al., Anomalous behaviors of visible luminescence from graphene quantum dots: interplay between size and shape, ACS Nano 6 (2012) 82038208 with permission from the American Chemical Society, © 2012.

triplet states in carbonyl groups (CQO) on sp2-carbon. Similarly, Li et al. synthesized N-doped CDs from HTC of folic acid, which demonstrated phosphorescence mainly due to the presence of CQN bonds promoting triplet states (Fig. 177) [42]. Likewise, the phosphorescence was greatly enhanced when the CDs were thermally embedded in a urea matrix due to the stabilizing effect. In addition, the authors demonstrated the deleterious effect of hydrogen bonding on phosphorescence by adding biuret which formed hydrogen bonds with CQN groups on CDs.

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FIGURE 17–6 GQDs synthesized from pyrolysis of GO demonstrated both (A) typical PL from UV excitation and (B) UCPL from visible range excitation. GO, graphene oxide; GQDs, graphene quantum dots; PL, photoluminescence; UCPL, upconversion photoluminescence. Reproduced from S. Zhuo, M. Shao, S.-T. Lee, Upconversion and downconversion fluorescent graphene quantum dots: ultrasonic preparation and photocatalysis, ACS Nano 6 (2012) 10591064 with permission from the American Chemical Society, © 2012.

17.5 Biomedical Applications of Carbon Dots 17.5.1 Biocompatibility and Bioimaging of Carbon Dots Nanomedicine has emerged as one of future paradigms of medicine [43]. The progress in nanotechnology for biomedical applications has exploded in the last two decades, with several nanomaterial-based therapeutics already in the market, and many more in clinical trials. One key area of nanomedicine is bioimaging, in which nanomaterials with optical properties are used as imaging probes and contrasting agents for various medical imaging techniques, such as magnetic resonance imaging (MRI) and computed tomography (CT) [44,45]. For example, superparamagnetic iron-oxide nanoparticles (commonly termed “SPIONs”) are commercialized and widely used as contrast agents for MRI. 18F-labeled molecules (e.g., florbetapir, flutemetamol) are used for positron emission tomography in amyloid detection in Alzheimer’s disease. CDs, regardless of their type, have been obviously considered prime candidates as probes for various medical imaging tools from the beginning for their tunable PL, especially for X-ray fluorescent CT. Rather than attaching molecular fluorophores [e.g., fluorescein, rhodamine, green fluorescent protein (GFP)] to other nanoparticles, which require additional processing steps, CDs with inherent PL properties, combined with inexpensive and simple fabrication, are considered a highly attractive choice. More significantly, CDs made mostly of carbon with a hydrophilic surface have been shown to demonstrate excellent biocompatibility both in vitro and in vivo, especially compared to heavy metal-based SQDs whose potential biomedical applications have long been scrapped due to severe cytotoxicity, despite generally possessing higher quantum yield and more robust emission spectra than CDs. Yang et al. first demonstrated the imaging of CDs injected into various tissue in mice models,

FIGURE 17–7 Phosphorescence of N-doped CDs having C 5QN bonds embedded in a urea matrix. The matrix stabilized the triplet states of C 5QN bonds and prevented their nonradiative relaxation via hydrogen bonding. The addition of biuret to induce hydrogen bonds reduced the phosphorescence intensity and lifetime. CDs, carbon dots. Reproduced from Q. Li, M. Zhou, Q. Yang, Q. Wu, J. Shi, A. Gong, et al., Efficient room-temperature phosphorescence from nitrogen-doped carbon dots in composite matrices, Chem. Mater. 28 (2016) 82218227 with permission from the American Chemical Society, © 2016.

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which showed strong PL properties and biocompatibility [46]. Detailed toxicology, biodistribution, and pharmacokinetics of CDs have been studied, which demonstrated a biodistribution pattern similar to other nanomaterials previously reported in the literature (e.g., greater accumulation in the liver, spleen, and kidney), gradual clearance from the body, and did not show any meaningful sign of blood or tissue toxicity (Fig. 178) [47,48]. Several cytotoxicity studies using various in vitro cell cultures have also demonstrated that CDs are generally nontoxic up to concentrations much higher than typical dosages for imaging probes. Wang et al. demonstrated biocompatibility of CQDs synthesized by hydrothermal treatment of polypyrrole nanoparticles in nitric acid solution in two different cell lines, MCF-7 and HT-29, in which cell viability was well maintained up to 0.1 mg/mL [49]. The biocompatibility was dependent on the type of surface passivating agent; poly(ethylene glycol) was more effective in enhancing the biocompatibility of CQDs than amine-based polymers, likely due to the cell membrane disruption by the increased positive charge. Similarly, GQDs conjugated with poly(ethylene glycol) showed excellent in vitro biocompatibility, up to 0.16 mg/mL for HeLa cells, as demonstrated by Chong et al. (Fig. 179) [50]. CDs are often conjugated with additional imaging modalities for multimode imaging probes to take advantage of their inherent PL properties as well as an avenue for chemical modifications. The most common approach is to incorporate conventional contrast agents to CDs. For example, SPIONs and Gd(III) have been frequently adopted to impart CDs with MRI contrasting as well as PL imaging capabilities [51,52].

17.5.2 Carbon Dots as Biosensors The primary application of CDs in biomedicine is biosensing, due to several appealing attributes: aqueous solubility, biocompatibility, tunable PL, and considerable photostability and quantum yield. CDs, as synthesized, do not possess any functionalities that could specifically interact with a biological molecule. Therefore, in order to be utilized as sensors, CDs should be decorated with targeting moieties that can interact with biomolecules of interest, and as a result it must accompany a discernible change in PL properties. For example, CQD modified with boronic acid (B(OH)2) was developed, which showed highly sensitive change in PL behavior in response to glucose. Polyamine-functionalized CDs, which retain positive charge under aqueous conditions and could act as chelators, have been utilized for metal ion detection [53,54]. A more traditional biosensing mechanism utilizing antibodies and aptamers has been incorporated to CDs as well. Ma et al. synthesized amine-functionalized CDs chemically immobilized with either antibody or aptamer against mucin 1 protein (MUC1) using glutaraldehyde as a crosslinker, and demonstrated the highly sensitive dose-dependent PL response against MUC1 in the nanomolar range (Fig. 1710A) [55]. With many CDs containing a significant portion of sp2-carbons, they have been shown to display high affinity toward DNA via ππ stacking. Also, CDs can be easily modified with positively charged amine groups via surface passivation, which allows for the electrostatic interaction with DNA. For example, Maiti et al. developed hybrid nanostructures consisting of double-stranded DNA adsorbed onto quaternized CNDs synthesized from betaine and

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FIGURE 17–8 In vivo evaluation of CDs using a rat model. (A) Histological analyses of different tissues (20 mg/kg body weight at 30 days). (B) Blood hematology analyses (0.2, 2, 20 mg/kg body weight up to 28 days). CDs, carbon dots. Reproduced from K. Wang, Z. Gao, G. Gao, Y. Wo, Y. Wang, G. Shen, et al., Systematic safety evaluation on photoluminescent carbon dots, Nanoscale Res Lett 8 (2013) 122 with permission from Springer, © 2013.

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FIGURE 17–9 In vitro evaluation of GQDs by measuring (A) viability, (B) percentages of cells in different viability stages, (C) LDH content, and (D) reactive oxygen species of HeLa cells after GQD treatment. GQDs, graphene quantum dots. LDH, lactate dehydrogenase. Reproduced from Y. Chong, Y. Ma, H. Shen, X. Tu, X. Zhou, J. Xu, et al., The in vitro and in vivo toxicity of graphene quantum dots, Biomaterials 35 (2014) 50415048 with permission from Elsevier, © 2014.

Tris via pyrolysis [56]. The DNACND nanohybrid materials showed highly specific binding affinity toward histone, resulting in PL recovery upon binding, which demonstrated their potential as a biosensor. Loo et al. similarly developed CQDs physically conjugated with fluorescently labeled ssDNA. The PL of the ssDNA was quenched when bound to the CQDs, but binding with the target complementary ssDNA led to the detachment from the CQDs and the recovery of PL in a dose-dependent manner (Fig. 1710B) [57]. The fluorescence resonance energy transfer (FRET) technique is increasingly investigated as a mode of biosensors. With two chromophores in close proximity (typically within 10 nm), the excitation of one chromophore (“donor”) and the energy is subsequently transferred to the other chromophore (“acceptor”) via nonradiative dipoledipole coupling. Therefore, a binding phenomenon between the sensor and the ligand having chromophores can be easily detected by measuring the transferred energy in the form of PL of the acceptor. With the inherent PL properties of CDs, it has the advantage of bypassing a chromophore conjugation step for either the donor or acceptor. Tang et al. first reported the synthesis of CDs loaded with anticancer drug, doxorubicin (DOX), in which the excitation of CDs at 405 nm resulted

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FIGURE 17–10 (A) Aptamer- and antibody-presenting CDs interacting with target protein (MUC1) resulted in decreased PL intensity. (B) Fluorescent single-stranded DNA (ssDNA) (FAM-Lprobe) was quenched when bound to CQDs, but detached from CQDs and recovered the fluorescence upon binding with the target complementary DNA. CDs, carbon dots; CQDs, carbon quantum dots; PL, photoluminescence. (A) Reproduced from N. Ma, W. Jiang, T. Li, Z. Zhang, H. Qi, M. Yang, Fluorescence aggregation assay for the protein biomarker mucin 1 using carbon dotlabeled antibodies and aptamers, Microchim. Acta 182 (2015) 443447 with permission from Springer-Verlag Wien, © 2014. (B) Reproduced from N. Ma, W. Jiang, T. Li, Z. Zhang, H. Qi, M. Yang, Fluorescence aggregation assay for the protein biomarker mucin 1 using carbon dot-labeled antibodies and aptamers, Microchim. Acta 182 (2015) 443447 with permission from the American Chemical Society, © 2016.

in the emission at 595 nm by DOX due to FRET from CD, in addition to the CD emission at 498 nm [58]. With the release of DOX and the gradual loss of FRET, the emission intensity at 498 nm increased and that at 595 nm decreased, allowing for the simultaneous monitoring of drug release. Dai et al. similarly deployed CQDs as FRET donors to detect the melamine content, a common cause of food poisoning (Fig. 1711) [59]. Here, gold nanoparticles (AuNPs) as FRET acceptors quenched the PL from CQDs, but the presence of melamine prevented the interaction between CQDs and AuNPs, and thereby blocked the FRET resulting in the recovery of PL intensity in a dose-dependent manner.

17.5.3 Theranostic Carbon Dots As the latest research trend in nanomedicine, theranostics, as the name indicates, aims to achieve the ultimate goal of medical breakthrough by combining the therapeutic and diagnostic functionalities in a single platform [60]. It requires intense engineering strategies to impart multiple functions to a material in nanometer scale. In many cases, the theranostic nanomedicine involves the attachment of imaging modalities such as radioactive (e.g., 125I, 18F)

FIGURE 17–11 (A) The PL of CQDs (donor) was quenched by FRET to the bound AuNPs (acceptor). (B) Upon the addition of melamine which binds to the CQDs, the AuNPs were released and the PL of CQDs was recovered. CQDs, carbon quantum dots; FRET, fluorescence resonance energy transfer; PL, photoluminescence. Reproduced from H. Dai, Y. Shi, Y. Wang, Y. Sun, J. Hu, P. Ni, et al., A carbon dot based biosensor for melamine detection by fluorescence resonance energy transfer, Sens. Actuators B: Chem. 202 (2014) 201208 with permission from Elsevier, © 2014.

FIGURE 17–12 (A) Schematic illustration of theranostic CND conjugated with cisplatin prodrug encapsulated in negatively charged polymeric nanocarriers. In an acidic tumor microenvironment, the nanocarriers would disintegrate and release the CNDcisplatin prodrug which would enter the tumor cells. The cisplatin is then released from the CND and exerts an antitumor effect. (B) The PL of CNDs was recovered within tumor cells only under acidic condition. (C) The antitumor effect of the theranostic CNDs was demonstrated both in in vitro and in vivo models. CNDs, carbon nanodots; PL, photoluminescence. Reproduced from T. Feng, X. Ai, G. An, P. Yang, Y. Zhao, Charge-convertible carbon dots for imaging-guided drug delivery with enhanced in vivo cancer therapeutic efficiency, ACS Nano 10 (2016) 44104420 with permission from the American Chemical Society, © 2016.

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and luminescent (e.g., indocyanine, Alexa Fluor, GFP) probes to drug nanocarriers. For nanomaterials with unique optical properties, such as AuNPs and SPIONs, that can be directly utilized as imaging probes as is, their therapeutic functions are provided by coorganizing with drug carriers or simply attaching drug molecules on the surface. Carbon nanomaterials such as CNTs, GO, and nanodiamonds have also been utilized as theranostic nanomaterials due to their inherent optical properties (e.g., NIR fluorescence, Raman scattering) as well as photothermic properties ideal for photodynamic anticancer therapy [61,62]. CDs with their PL properties are no stranger to theranostic nanomedicine. In addition to their PL properties, there are several doping techniques to functionalize CDs with heterogeneous chemical structures. In addition, most CDs contain hydrophilic functional groups such as carbonyl and hydroxyl groups that can be further modified to attach various functional moieties. For example, Zheng et al. developed cancer theranostic CQDs by covalently conjugating oxaliplatin, a well-known cancer therapeutic, to the surface of amine-functionalized CQDs via amide coupling [63]. The oxaliplatin-linked CQDs showed anticancer activity and allowed for multiphoton imaging using an in vitro model. Alternatively, Feng et al. developed larger, stimuli-responsive, self-assembled nanocarriers encapsulated with CNDs conjugated with cisplatin prodrug (Fig. 1712) [64]. With the negatively charged nanocarriers, the acidic tumor environment led to their structural disintegration and subsequent release of the cisplatin-loaded CNDs which demonstrated simultaneous PL imaging and antitumor activity. CDs with a larger sp2 carbon content or conjugated electron system have significant absorbance at the NIR region and display photothermal activities, similar to graphene and CNT. Therefore, theranostic CDs capable of photodynamic therapies (PDTs) are also being developed. Ge et al. synthesized CNDs from hydrothermal pyrolysis of a conductive pheylpropanoic acid-substituted polythiophene, which showed enhanced red emission (from 500 to 800 nm) and photothermal conversion efficiency (above 35%) [65]. They also demonstrated the multifunctionality of these CNDs using an in vivo model; PL, photoacoustic imaging, and PDT. It has been shown that the photothermal effects of CDs could be further enhanced by conjugating photosensitizers to increase the light absorption [66].

17.6 Conclusion With many attributes associated with CDs (e.g., facile synthesis, tunable PL, physicochemical modifications, aqueous solubility, biocompatibility), especially compared to other carbonbased nanomaterials such as CNTs, GO, and nanodiamonds, the scientific progress of CD technology despite its short history has been nothing short of remarkable, especially within the last decade. As covered in this chapter, the development of a diverse array of CD-based nanomedicines is already underway, with their enormous potential for biomedical applications. Therefore, taking everything into account, this research trend is expected to continue in the near future. Like other “nanobiomaterials” that came before, the clinical translation of CDs will hinge on their systematic biological evaluation (e.g., in vitro and in vivo toxicology, pharmacokinetics, efficacy). Furthermore, due to the versatility and diversity of CDs, it is

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imperative that the standardization of CDs, in terms of synthetic methodology and physicochemical properties, should be a priority moving forward for clinical translation. Since this is a highly active area of research, readers are encouraged to follow up on the latest research outcome on CDs.

References [1] J.R. McCarthy, R. Weissleder, Multifunctional magnetic nanoparticles for targeted imaging and therapy, Adv. Drug Deliv. Rev. 60 (2008) 12411251. [2] C.J. Murphy, A.M. Gole, J.W. Stone, P.N. Sisco, A.M. Alkilany, E.C. Goldsmith, et al., Gold nanoparticles in biology: beyond toxicity to cellular imaging, Acc. Chem. Res. 41 (2008) 17211730. [3] W. Xie, P. Qiu, C. Mao, Bio-imaging, detection and analysis by using nanostructures as SERS substrates, J. Mater. Chem. 21 (2011) 51905202. [4] G. Hong, S. Diao, A.L. Antaris, H. Dai, Carbon nanomaterials for biological imaging and nanomedicinal therapy, Chem. Rev. 115 (2015) 1081610906. [5] F. Pinaud, X. Michalet, L.A. Bentolila, J.M. Tsay, S. Doose, J.J. Li, et al., Advances in fluorescence imaging with quantum dot bio-probes, Biomaterials 27 (2006) 16791687. [6] X. Xu, R. Ray, Y. Gu, H.J. Ploehn, L. Gearheart, K. Raker, et al., Electrophoretic analysis and purification of fluorescent single-walled carbon nanotube fragments, J. Am. Chem. Soc. 126 (2004) 1273612737. [7] P. Zuo, X. Lu, Z. Sun, Y. Guo, H. He, A review on syntheses, properties, characterization and bioanalytical applications of fluorescent carbon dots, Microchim. Acta 183 (2016) 519542. [8] Y. Wang, A. Hu, Carbon quantum dots: synthesis, properties and applications, J. Mater. Chem. C 2 (2014) 69216939. [9] S.Y. Lim, W. Shen, Z. Gao, Carbon quantum dots and their applications, Chem. Soc. Rev. 44 (2015) 362381. [10] R. Hardman, A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors, Environ. Health Perspect. 114 (2006) 165172. [11] A. Cayuela, M.L. Soriano, C. Carrillo-Carrión, M. Valcárcel, Semiconductor and carbon-based fluorescent nanodots: the need for consistency, Chem. Commun. 52 (2016) 13111326. [12] S. Zhu, Y. Song, X. Zhao, J. Shao, J. Zhang, B. Yang, The photoluminescence mechanism in carbon dots (graphene quantum dots, carbon nanodots, and polymer dots): current state and future perspective, Nano Res. 8 (2015) 355381. [13] S. Zhu, Y. Song, J. Shao, X. Zhao, B. Yang, Non-conjugated polymer dots with crosslink-enhanced emission in the absence of fluorophore units, Angew. Chem. Int. Ed. 54 (2015) 1462614637. [14] L.R. Radovic, B. Bockrath, On the chemical nature of graphene edges: origin of stability and potential for magnetism in carbon materials, J. Am. Chem. Soc. 127 (2005) 59175927. [15] D. Pan, J. Zhang, Z. Li, M. Wu, Hydrothermal route for cutting graphene sheets into blue-luminescent graphene quantum dots, Adv. Mater. 22 (2010) 734738. [16] L. Lin, S. Zhang, Creating high yield water soluble luminescent graphene quantum dots via exfoliating and disintegrating carbon nanotubes and graphite flakes, Chem. Commun. 48 (2012) 1017710179. [17] H. Zeng, X.-W. Du, S.C. Singh, S.A. Kulinich, S. Yang, J. He, et al., Nanomaterials via laser ablation/irradiation in liquid: a review, Adv. Funct. Mater. 22 (2012) 13331353. [18] H.W. Kroto, J.R. Heath, S.C. O'Brien, R.F. Curl, R.E. Smalley, C60: buckminsterfullerene, Nature 318 (1985) 162.

398

THERANOSTIC BIONANOMATERIALS

[19] C.D. Scott, S. Arepalli, P. Nikolaev, R.E. Smalley, Growth mechanisms for single-wall carbon nanotubes in a laser-ablation process, Appl. Phys. A 72 (2001) 573580. [20] Y.-P. Sun, B. Zhou, Y. Lin, W. Wang, K.A.S. Fernando, P. Pathak, et al., Quantum-sized carbon dots for bright and colorful photoluminescence, J. Am. Chem. Soc. 128 (2006) 77567757. [21] X. Li, H. Wang, Y. Shimizu, A. Pyatenko, K. Kawaguchi, N. Koshizaki, Preparation of carbon quantum dots with tunable photoluminescence by rapid laser passivation in ordinary organic solvents, Chem. Commun. 47 (2011) 932934. [22] S. Hu, J. Liu, J. Yang, Y. Wang, S. Cao, Laser synthesis and size tailor of carbon quantum dots, J. Nanopart. Res. 13 (2011) 72477252. [23] M. Bottini, C. Balasubramanian, M.I. Dawson, A. Bergamaschi, S. Bellucci, T. Mustelin, Isolation and characterization of fluorescent nanoparticles from pristine and oxidized electric arc-produced singlewalled carbon nanotubes, J. Phys. Chem. B 110 (2006) 831836. [24] J. Lu, J.-X. Yang, J. Wang, A. Lim, S. Wang, K.P. Loh, One-pot synthesis of fluorescent carbon nanoribbons, nanoparticles, and graphene by the exfoliation of graphite in ionic liquids, ACS Nano 3 (2009) 23672375. [25] X. Tan, Y. Li, X. Li, S. Zhou, L. Fan, S. Yang, Electrochemical synthesis of small-sized red fluorescent graphene quantum dots as a bioimaging platform, Chem. Commun. 51 (2015) 25442546. [26] V. Georgakilas, J.A. Perman, J. Tucek, R. Zboril, Broad family of carbon nanoallotropes: classification, chemistry, and applications of fullerenes, carbon dots, nanotubes, graphene, nanodiamonds, and combined superstructures, Chem. Rev. 115 (2015) 47444822. [27] F. Wang, S. Pang, L. Wang, Q. Li, M. Kreiter, C.-y Liu, One-step synthesis of highly luminescent carbon dots in noncoordinating solvents, Chem. Mater. 22 (2010) 45284530. [28] S. Zhu, J. Zhang, C. Qiao, S. Tang, Y. Li, W. Yuan, et al., Strongly green-photoluminescent graphene quantum dots for bioimaging applications, Chem. Commun. 47 (2011) 68586860. [29] P. Atienzar, A. Primo, C. Lavorato, R. Molinari, H. García, Preparation of graphene quantum dots from pyrolyzed alginate, Langmuir 29 (2013) 61416146. [30] D. Chowdhury, N. Gogoi, G. Majumdar, Fluorescent carbon dots obtained from chitosan gel, RSC Adv. 2 (2012) 1215612159. [31] Z.-C. Yang, M. Wang, A.M. Yong, S.Y. Wong, X.-H. Zhang, H. Tan, et al., Intrinsically fluorescent carbon dots with tunable emission derived from hydrothermal treatment of glucose in the presence of monopotassium phosphate, Chem. Commun. 47 (2011) 1161511617. [32] B. Hu, K. Wang, L. Wu, S.-H. Yu, M. Antonietti, M.-M. Titirici, Engineering carbon materials from the hydrothermal carbonization process of biomass, Adv. Mater. 22 (2010) 813828. [33] H. Zhu, X. Wang, Y. Li, Z. Wang, F. Yang, X. Yang, Microwave synthesis of fluorescent carbon nanoparticles with electrochemiluminescence properties, Chem. Commun. (2009) 51185120. [34] X. Zhai, P. Zhang, C. Liu, T. Bai, W. Li, L. Dai, et al., Highly luminescent carbon nanodots by microwave-assisted pyrolysis, Chem. Commun. 48 (2012) 79557957. [35] H. Li, X. He, Y. Liu, H. Yu, Z. Kang, S.-T. Lee, Synthesis of fluorescent carbon nanoparticles directly from active carbon via a one-step ultrasonic treatment, Mater. Res. Bull. 46 (2011) 147151. [36] J. Zong, Y. Zhu, X. Yang, J. Shen, C. Li, Synthesis of photoluminescent carbogenic dots using mesoporous silica spheres as nanoreactors, Chem. Commun. 47 (2011) 764766. [37] S. Bhattacharyya, B. Konkena, K. Jayaramulu, W. Schuhmann, T.K. Maji, Synthesis of nano-porous carbon and nitrogen doped carbon dots from an anionic MOF: a trace cobalt metal residue in carbon dots promotes electrocatalytic ORR activity, J. Mater. Chem. A 5 (2017) 1357313580. [38] S. Kim, S.W. Hwang, M.-K. Kim, D.Y. Shin, D.H. Shin, C.O. Kim, et al., Anomalous behaviors of visible luminescence from graphene quantum dots: interplay between size and shape, ACS Nano 6 (2012) 82038208.

Chapter 17 • Multimodal Carbon Dots as Biosensors

399

[39] S. Zhuo, M. Shao, S.-T. Lee, Upconversion and downconversion fluorescent graphene quantum dots: ultrasonic preparation and photocatalysis, ACS Nano 6 (2012) 10591064. [40] X. Jia, J. Li, E. Wang, One-pot green synthesis of optically pH-sensitive carbon dots with upconversion luminescence, Nanoscale 4 (2012) 55725575. [41] Y. Deng, D. Zhao, X. Chen, F. Wang, H. Song, D. Shen, Long lifetime pure organic phosphorescence based on water soluble carbon dots, Chem. Commun. 49 (2013) 57515753. [42] Q. Li, M. Zhou, Q. Yang, Q. Wu, J. Shi, A. Gong, et al., Efficient room-temperature phosphorescence from nitrogen-doped carbon dots in composite matrices, Chem. Mater. 28 (2016) 82218227. [43] D.F. Emerich, C.G. Thanos, Nanotechnology and medicine, Expert Opin. Biol. Ther. 3 (2003) 655663. [44] M.A. Hahn, A.K. Singh, P. Sharma, S.C. Brown, B.M. Moudgil, Nanoparticles as contrast agents for invivo bioimaging: current status and future perspectives, Anal. Bioanal. Chem. 399 (2011) 327. [45] H. Koo, M.S. Huh, J.H. Ryu, D.-E. Lee, I.-C. Sun, K. Choi, et al., Nanoprobes for biomedical imaging in living systems, Nano Today 6 (2011) 204220. [46] S.-T. Yang, L. Cao, P.G. Luo, F. Lu, X. Wang, H. Wang, et al., Carbon dots for optical imaging in vivo, J. Am. Chem. Soc. 131 (2009) 1130811309. [47] H. Tao, K. Yang, Z. Ma, J. Wan, Y. Zhang, Z. Kang, et al., In vivo NIR fluorescence imaging, biodistribution, and toxicology of photoluminescent carbon dots produced from carbon nanotubes and graphite, Small 8 (2012) 281290. [48] K. Wang, Z. Gao, G. Gao, Y. Wo, Y. Wang, G. Shen, et al., Systematic safety evaluation on photoluminescent carbon dots, Nanoscale Res. Lett. 8 (2013) 122. [49] Y. Wang, P. Anilkumar, L. Cao, J.-H. Liu, P.G. Luo, K.N. Tackett, et al., Carbon dots of different composition and surface functionalization: cytotoxicity issues relevant to fluorescence cell imaging, Exp. Biol. Med. 236 (2011) 12311238. [50] Y. Chong, Y. Ma, H. Shen, X. Tu, X. Zhou, J. Xu, et al., The in vitro and in vivo toxicity of graphene quantum dots, Biomaterials 35 (2014) 50415048. [51] H. Wang, J. Shen, Y. Li, Z. Wei, G. Cao, Z. Gai, et al., Magnetic iron oxidefluorescent carbon dots integrated nanoparticles for dual-modal imaging, near-infrared light-responsive drug carrier and photothermal therapy, Biomater. Sci. 2 (2014) 915923. [52] H. Yang, S. Santra, G.A. Walter, P.H. Holloway, GdIII-functionalized fluorescent quantum dots as multimodal imaging probes, Adv. Mater. 18 (2006) 28902894. [53] K. Qu, J. Wang, J. Ren, X. Qu, Carbon dots prepared by hydrothermal treatment of dopamine as an effective fluorescent sensing platform for the label-free detection of iron(III) ions and dopamine, Chem. Eur. J. 19 (2013) 72437249. [54] Y. Dong, R. Wang, G. Li, C. Chen, Y. Chi, G. Chen, Polyamine-functionalized carbon quantum dots as fluorescent probes for selective and sensitive detection of copper ions, Anal. Chem. 84 (2012) 62206224. [55] N. Ma, W. Jiang, T. Li, Z. Zhang, H. Qi, M. Yang, Fluorescence aggregation assay for the protein biomarker mucin 1 using carbon dot-labeled antibodies and aptamers, Microchim. Acta 182 (2015) 443447. [56] S. Maiti, K. Das, P.K. Das, Label-free fluorimetric detection of histone using quaternized carbon dotDNA nanobiohybrid, Chem. Commun. 49 (2013) 88518853. [57] A.H. Loo, Z. Sofer, D. Bouˇsa, P. Ulbrich, A. Bonanni, M. Pumera, Carboxylic carbon quantum dots as a fluorescent sensing platform for DNA detection, ACS Appl. Mater. Interfaces 8 (2016) 19511957. [58] J. Tang, B. Kong, H. Wu, M. Xu, Y. Wang, et al., Carbon nanodots featuring efficient FRET for real-time monitoring of drug delivery and two-photon imaging, Adv. Mater. 25 (2013) 65696574.

400

THERANOSTIC BIONANOMATERIALS

[59] H. Dai, Y. Shi, Y. Wang, Y. Sun, J. Hu, P. Ni, et al., A carbon dot based biosensor for melamine detection by fluorescence resonance energy transfer, Sens. Actuators B: Chem. 202 (2014) 201208. [60] S.M. Janib, A.S. Moses, J.A. MacKay, Imaging and drug delivery using theranostic nanoparticles, Adv. Drug Deliv. Rev. 62 (2010) 10521063. [61] C. Cha, S.R. Shin, N. Annabi, M.R. Dokmeci, A. Khademhosseini, Carbon-based nanomaterials: multifunctional materials for biomedical engineering, ACS Nano 7 (2013) 28912897. [62] M. Kim, J. Jang, C. Cha, Carbon nanomaterials as versatile platforms for theranostic applications, Drug Discov. Today 22 (2017) 14301437. [63] M. Zheng, S. Liu, J. Li, D. Qu, H. Zhao, X. Guan, et al., Integrating oxaliplatin with highly luminescent carbon dots: an unprecedented theranostic agent for personalized medicine, Adv. Mater. 26 (2014) 35543560. [64] T. Feng, X. Ai, G. An, P. Yang, Y. Zhao, Charge-convertible carbon dots for imaging-guided drug delivery with enhanced in vivo cancer therapeutic efficiency, ACS Nano 10 (2016) 44104420. [65] J. Ge, Q. Jia, W. Liu, L. Guo, Q. Liu, M. Lan, et al., Red-emissive carbon dots for fluorescent, photoacoustic, and thermal theranostics in living mice, Adv. Mater. 27 (2015) 41694177. [66] P. Huang, J. Lin, X. Wang, Z. Wang, C. Zhang, M. He, et al., Light-triggered theranostics based on photosensitizer-conjugated carbon dots for simultaneous enhanced-fluorescence imaging and photodynamic therapy, Adv. Mater. 24 (2012) 51045110.

18 Bionanomaterials as Imaging Contrast Agents Xia Cao1,2, Di Huang1,3, Yu Shrike Zhang1 1

DIVISION OF ENGINEERI NG IN MEDICINE, DEPART ME NT OF ME DICINE, BRIGHAM AND WOM EN’S HOSPITAL, HARVARD MEDICAL SCHOOL, C AMBRIDGE, MA, UNITED STATES 2 DEPARTME NT OF PHARMACEUTICS AND TISSUE ENGINEERING, SCHOOL OF P H AR M AC Y , JI AN G S U U NIV E R S I T Y, Z HE N J I A NG , P . R . C H I NA 3 DEPART ME NT OF BIOMEDICAL ENGINEERING, RESEARCH CENTER FOR NANO-BIOMATERIALS AND REGENERATIVE MEDICINE, C OLLEGE OF BIO ME DI C A L E N G I N E E RI N G , T A I Y UA N UNIVERSITY OF TECHNOLOGY, TAIYUAN, P.R. CHINA

18.1 Introduction The development of imaging contrast agents provides great convenience both in clinical diagnostics and as a research tool to assist visualization in real time. The fundamental goal of noninvasive medical imaging is to detect and locate the target, pathway, or physiological function of the designated molecules in a tissue or organ. Recently, bionanomaterials have led to rapid development of new in vivo imaging technologies, and the application of bionanomaterials in imaging strategies not only improves the sensitivity and specificity of diagnosis but also promotes our understanding on the safety of these materials, making them play a pivotal role in molecular imaging. Contrast agents derived from bionanomaterials may afford tight control over delivery and substantially increased diagnostic sensitivity and specificity than conventional approaches. Bionanomaterials as imaging contrast agents can be divided into several major categories depending on the imaging modality, such as magnetic bionanomaterials, optical bionanomaterials, acoustic bionanomaterials, and nuclear bionanomaterials, which are discussed in this chapter.

18.2 Magnetic Contrast-Enhancing Bionanomaterials Iron (Fe)-based and manganese (Mn)-based magnetic bionanomaterials are the most widely used magnetic bionanomaterials for applications in magnetic resonance imaging (MRI), magnetic particle imaging (MPI), and magnetomotive imaging, as shown in Fig. 18 1.

Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00018-3 © 2019 Elsevier Inc. All rights reserved.

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FIGURE 18–1 Types of magnetic contrast-enhancing bionanomaterials.

18.2.1 Magnetic Resonance Imaging MRI is a powerful tool for deep-penetration and high-quality volumetric imaging of tissues with anatomical details [1], which is based on magnetization where the changes in the direction of the rotational axis of protons found in water that makes up the living tissues are commonly used for forming images [2]. However, if one desires molecular information, contrast agents such as those based on bionanomaterials must be used in part as a result of its relatively low sensitivity. Many MRI contrast agents exist, which can be divided into two categories. T1 (longitudinal relaxation time) is the time constant that determines the rate at which excited protons return to equilibrium [3]. It is a measure of the time taken for spinning protons to realign with the external magnetic field. T2 (transverse relaxation time) is the time constant that determines the rate at which excited protons reach equilibrium or go out of phase with each other [4]. It is a measure of the time taken for the spinning protons to lose phase coherence among the nuclei spinning perpendicular to the main field. Some magnetic nanomaterials have been investigated to reinforce the T1 contrast, such as manganese oxide (MnO) nanomaterials. The reinforced contrast effects can be attained by controlling the size and morphology of the bionanomaterials. For instance, 5-nm Mn-based nanomaterials yielded high T1 contrast (and no T2 contrast), while 12-nm Mn-based nanomaterials displayed the inverse (high T2 contrast and no T1 contrast); 7-nm Mn-based nanomaterials were a good compromise if dual-mode T1/T2 MRI contrast agents are desired [5]. Also, reinforced T1 contrast can be achieved by modifying or controlling the specific morphology of the bionanomaterials. Hyeon and colleagues varied the size of the AS1411 (an aptamer showing specific binding to nucleolin)-polyethylene glycol (PEG)-MnO nanomaterials to optimize their contrast performances (Fig. 18 2) [6]. They applied surfactants as a structure-directing agent to control the MnO crystal growth in two dimensions (2D). After injection of the 2D nanoplates into mice, liver, spleen, and kidney uptakes were observed on T1-weighted images (Fig. 18 3). Meanwhile, by far the most commonly used nanomaterial for T2 contrast enhancement has been iron oxide (Fe3O4), as this class of bionanomaterial displays a high magnetization that can induce magnetic inhomogeneity that influences T2 relaxation [9]. In particular, Fe3O4 nanomaterials serve as T2 contrast agents as a function of the local magnetic field gradients they produce for their dipoles to interact with water protons [10]. Recently, activatable or “smart” Fe3O4 nanomaterials have been explored that take advantage of the fact that water molecules must be in the immediate proximity for them to function as T2 contrast agents. For

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FIGURE 18–2 (A) TEM images of water-dispersible AS1411-PEG-MnO nanoparticles with particle sizes of 7, 15, 20, and 25 nm. (B) T1-weighted MRI image of MnO nanoparticles from a 3.0-T clinical MRI system. MnO, Manganese oxide; MRI, magnetic resonance imaging; PEG, polyethylene glycol; TEM, transmission electron microscopy. Reproduced with permission from H.B. Na, J.H. Lee, K. An, Y.I. Park, M. Park, I.S. Lee, et al., Development of a T1 contrast agent for magnetic resonance imaging using MnO nanoparticles, Angew. Chem. Int. Ed. Engl. 46 (28) (2007) 5397 5401 [7].

FIGURE 18–3 Pseudo-color MRI T1 images of mice bearing renal carcinoma pre- and postinjection of the AS1411PEG-MnO nanoprobe. The red, yellow, and green arrows indicate tumor, heart, and liver, respectively. MnO, Manganese oxide; MRI, magnetic resonance imaging; PEG, polyethylene glycol. Reproduced with permission from J. Li, C. Wu, P. Hou, M. Zhang, K. Xu, One-pot preparation of hydrophilic manganese oxide nanoparticles as T1 nano-contrast agent for molecular magnetic resonance imaging of renal carcinoma in vitro and in vivo, Biosens. Bioelectron. 102 (2018) 1 8 [8].

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example, Fe3O4 nanomaterials (B6 nm in diameter) were constructed to be activated via copper-free click chemistry when exposed to matrix metalloproteases (MMPs) overexpressed in the tumor microenvironment [11 13]. Once exposed to MMPs to cleave the spacer, the two complementary Fe3O4 nanomaterials self-assembled into larger superparamagnetic nanoclusters that boosted T2 contrast by increasing the local magnetic susceptibility. Some researchers introduced a versatile strategy to design a smart nanoplatform using phase change material to encapsulate photosensitizer (zinc phthalocyanine, ZnPc) in copper sulfide-loaded Fe-doped tantalum oxide (Fe-m Ta2O5@CuS) nanoparticles [14]. The unique properties of Fe-m Ta2O5 endowed the nanoplatform with excellent computed tomography (CT) and T1-weighted MRI performance for guiding and real-time monitoring of the therapeutic effect. Another type of highly stable silica-coated manganese ferrite nanoparticle was synthesized for application as an MRI contrast agent [15]. The particle size was investigated using transmission electron microscopy (TEM) and was found to be 40 60 nm. The efficiency of the MRI contrast agents was investigated using aqueous solutions of the particles in a 4.7-T MRI scanner. The T1 and T2 relativities (r1 and r2) of the particles were 1.42 and 60.65 s/mM, respectively, in water. The ratio r2/r1 was 48.91, confirming that the silica-coated manganese ferrite nanoparticles were suitable high-efficacy T2 contrast agents.

18.2.2 Magnetic Particle Imaging MPI is an emerging noninvasive tomographic technique that directly detects superparamagnetic tracers. It is used in medical imaging to measure the three-dimensional (3D) localization and concentration of nanomaterials [16]. MPI systems use changing magnetic fields to generate signals from tracers such as superparamagnetic iron oxide (SPIO) nanoparticles; these fields are specifically designed to produce a single magnetic field-free region as signal, and an image is generated by moving this region across a sample [17]. Since there is no SPIO in the natural tissue, signals are only detected from the administered tracers achieving high sensitivity. Fe3O4 nanomaterials have been almost the only contrast agent applied for MPI thus far, because the underlying image-forming mechanism requires its nanomaterial tracers to display highly uniform sizes and surface properties. The physical properties of the superparamagnetic nanomaterials, including the size distribution, anisotropy of the magnetic core, and the surface modification, determine the spatial resolution and sensitivity of MPI imaging [18,19]. For example, PEG-coated spinel-phase Fe3O4 nanoparticles were prepared and studied as blood pool tracers for preclinical MPI (Fig. 18 4). The results indicated the optimized magnetic properties and long systemic retention of Fe3O4 nanoparticles coated with 20-kDa PEG, making it a promising blood pool MPI tracer, with potential to enable MPI imaging in cardio- and cerebrovascular diseases, and solid tumor diagnosis via the enhanced permeation and retention effect [20]. In addition to spinel phase Fe3O4, an aqueous synthesis method was presented for generating magnetic nanoparticles (magnetite/maghemite mixed-phase) with excellent magnetic characteristics, and therefore highly suited for both MRI and MPI [21].

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FIGURE 18–4 (A) TEM image of 25-nm SPIO nanoparticles and SAED confirming the spinel structure. (B) MPI subtraction images. APCmin/1, a mouse model genetically predisposed to GI polyp. GI, Gastrointestinal; MPI, magnetic particle imaging; SAED, small-angle electron diffraction; SPIO, superparamagnetic iron oxide; TEM, transmission electron microscopy. Reproduced with permission from E.Y. Yu, P. Chandrasekharan, R. Berzon, Z.W. Tay, X.Y. Zhou, A.P. Khandhar, et al., Magnetic particle imaging for highly sensitive, quantitative, and safe in vivo gut bleed detection in a murine model, ACS Nano 11 (12) (2017) 12067 12076 [22].

FIGURE 18–5 A schematic of the experimental setup used to measure pMMUS signal in a gelatin phantom with inclusions made from three different types of contrast agents with identical concentration (by metal mass). Inset shows TEM image of the Fe3O4 nanocluster-based MCA agent. MCA, Magnetoactive imaging contrast; pMMUS, pulsed magnetomotive ultrasound imaging; TEM, transmission electron microscopy. Reproduced with permission from M. Mehrmohammadi, T.H. Shin, M. Qu, P. Kruizinga, R.L. Truby, J.H. Lee, et al., In vivo pulsed magnetomotive ultrasound imaging using high-performance magnetoactive contrast nanoagents, Nanoscale 5 (22) (2013) 11179 11186 [27].

18.2.3 Magnetomotive Imaging Magnetomotive contrast agents are typically superparamagnetic bionanomaterials with high magnetic susceptibility that can be spatially shifted within a sample by a time-varying, external high magnetic field gradient [23]. Imaging is performed by instrumentation that is used to detect these minute positional shifts. To reinforce the contrast, Fe3O4 nanoclusters with enhanced magnetization were prepared and studied for pulsed magnetomotive ultrasound imaging (pMMUS) [24] (Fig. 18 5). The results indicated that by using the Fe3O4 nanoclusters with enhanced magnetic properties, the pMMUS signal increased significantly, which is

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an essential requirement for further development of in vivo pMMUS imaging [25]. Recently, a new generation of magnetomotive photoacoustic (mmPA) imaging featuring cyclic magnetic motion and ultrasound speckle tracking was developed. This advancement has enabled robust artifact elimination caused by physiologic motions and demonstrated the application of the mmPA technology for sensitive tumor imaging in vivo [26].

18.3 Optical Contrast-Enhancing Bionanomaterials Optical imaging is perhaps the most accessible form of imaging. Optical bionanomaterials can be divided into several categories targeted to enhance contrasts in luminescence, fluorescence resonance energy transfer (FRET), Raman imaging, optical coherence tomography (OCT), and photoacoustic imaging (PAI), shown in Fig. 18 6.

18.3.1 Luminescence Luminescence is an umbrella term for entities that emit light upon energetic excitation without heating. Luminescence bionanomaterials have broad applications, including those in optical devices such as photovoltaics [28], security [29], lighting [30], lasers [31], diagnostic sensing [32], color display [33], and for in vivo imaging [34]. Luminescence includes photoluminescence (such as fluorescence) [35], bioluminescence [36], chemiluminescence [37], phosphorescence [38], and mechanoluminescence [39], among others. Some research has reported iridium-coated gold nanoparticles as probes for multiphoton lifetime imaging with characteristic long luminescent lifetimes based on iridium luminescence in the range of hundreds of nanoseconds and a short signal on the scale of pico-seconds based on gold, allowing multichannel detection [40]. Others have developed a contrast agent, ZnGa2O4Cr0.004 (ZGC), used for guided surgery during operations for accurate delineation of hepatocellular carcinoma. ZGC showed excellent long-lasting afterglow properties that lasted for hours, which could aid in real-time visualization to facilitate guided surgery (Fig. 18 7). Meanwhile, ZGC displayed high spatial resolution and deep penetration during preoperation for diagnostic CT. This new multimodality nanoparticle has great potential for accurate liver cancer imaging and resection guidance [41].

18.3.2 Fluorescence Resonance Energy Transfer The most well-known and widely used form of optical resonance energy transfer (nonradioactive transfer of virtual photons from donor to a nearby acceptor) in imaging is FRET, which

FIGURE 18–6 Types of optical contrast-enhancing bionanomaterials.

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FIGURE 18–7 (A) Schematic illustration of the synthesis route of ZGC. (B) Biomedical applications of ZGC. ZGC, ZnGa2O4Cr0.004. Reproduced with permission from T. Ai, W. Shang, H. Yan, C. Zeng, K. Wang, Y. Gao, et al., Near infrared-emitting persistent luminescent nanoparticles for hepatocellular carcinoma imaging and luminescenceguided surgery, Biomaterials 167 (2018) 216 225 [42].

has been used, for instance, to monitor enzymatic activity within living subjects. FRET is a mechanism describing energy transfer between two light-sensitive molecules (chromophores) [43 45]. A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. Measurements of FRET efficiency can be used to determine if two fluorophores are within a certain distance of each other. Such measurements are used as a research tool in fields including biology and chemistry. FRET is analogous to near-field communication, in that the radius of interaction is much smaller than the wavelength of light emitted [46]. In the nearfield region, the excited chromophore emits a virtual photon that is instantly absorbed by a receiving chromophore. These virtual photons are undetectable, since their existence violates

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FIGURE 18–8 (A) Design of GQD 2 hydroIR783 nanoprobe for in situ, real-time, ratiometric fluorescence monitoring of O2•2 and •OH in vivo. (B) The reaction between O2•2 and •OH and the hydroIR783. (C) Normalized Abs and FL of GQDs and IR783 in phosphate-buffered saline (pH 5 7.4). Abs, Absorption spectra; FL, fluorescence spectra; GQD, graphene quantum dot. Reproduced with permission from R. Liu, L. Zhang, Y. Chen, Z. Huang, Y. Huang, S. Zhao, Design of a new near-infrared ratiometric fluorescent nanoprobe for real-time imaging of superoxide anions and hydroxyl radicals in live cells and in situ tracing of the inflammation process in vivo, Anal. Chem. 90 (7) (2018) 4452 4460 [51].

the conservation of energy and momentum, and hence FRET is known as a radiationless mechanism [47]. Quantum electrodynamical calculations have been used to determine that radiationless (FRET) and radiative energy transfer are the short- and long-range asymptotes of a single unified mechanism [48]. FRET requires input stimulation light. However, by intelligently pairing materials together, many groups have optically imaged tumors deep inside living animals without the need for excitation light and the resulting autofluorescence could achieve very high sensitivities [49,50]. For example, a FRET-based ratiometric fluorescent nanoprobe could monitor O2•2 and •OH generation in real time and trace the inflammation process in situ and in vivo. The proposed nanoprobe was composed of PEG-functionalized luminescent graphene quantum dots as the energy donor connecting to hydroIR783, serving as both the O2•2 and •OH recognizing ligand and the energy acceptor. The nanoprobe not only exhibited a fast response to O2•2 and •OH but also presented good biocompatibility as well as a high photostability and signal-to-noise ratio (Fig. 18 8).

18.3.3 Raman Raman imaging is a powerful technique for sample’s Raman spectrum. This technique and generates an infinitesimal amount of Raman spectrum using a camera [52]. The

generating detailed chemical images based on a uses a laser light source to irradiate a sample, Raman scattered light, which is detected as a characteristic fingerprinting pattern in a Raman

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spectrum makes it possible to identify substances including polymorphs and evaluate local crystallinity, orientation, and stress [53,54]. A complete spectrum is acquired at each and every pixel of the image, and then interrogated to generate false color images based on material composition, phase, crystallinity, and strain [55]. With its ultrahigh specificity, relatively high penetration depth, and high-dimensional multiplexing potential, Raman imaging is a viable alternative to fluorescent imaging. The feasibility of using plasmonic nanoparticlebased microscopy with both surface-enhanced Raman scattering spectroscopy (SERS) and dark-field spectroscopic imaging was demonstrated. This technique provided several advantages over conventional fluorescence labeling. The brightness of SERS signal and the absence of background allowed for short imaging times, and there was no degradation of the signal intensity over time and under high-power illumination [56]. To sensitively and quantitatively detect and image reactive oxygen species (ROS), some other research described the design and synthesis of myoglobin- and polydopamine-engineered SERS (MP-SERS) nanoprobes with strong, tunable SERS signals [57] (Fig. 18 9).

18.3.4 Optical Coherence Tomography OCT typically derives its distinctive contrast capabilities from the scattering properties of biological tissues such as cells, extracellular matrix molecules, and other stromal and vesicular components. OCT is based on low-coherence interferometry, typically employing nearinfrared light. The use of relatively long-wavelength light allows it to penetrate into the scattering medium [58]. Therefore, fundamentally, a critical aspect in the pursuit of appropriate bionanomaterials to generate basic OCT contrast is their scattering properties [59]. Recently, a real-time noninvasive imaging methodology was developed by integrating Fe3O4 nanoparticles into polymeric microneedles (MNs) to enhance image contrast for micro-OCT (µOCT) imaging. The results showed that a concentration of B4 5 wt.% Fe3O4 nanoparticles not only helped achieve the best contrast-to-noise ratio in µOCT imaging, which is about 10 times higher than that without Fe3O4 nanoparticles in air and hydrogel, but also enabled a clear observation of real-time changes in the profile of MNs in their swelling process in skin tissues [60]. Also, a platform of OCT that enabled high-resolution in vivo imaging and concomitant targeted therapy was developed to obtain wide-field in vivo imaging of nanoparticles [61]. The platform included the first in vivo images of nanoparticle pharmacokinetics acquired with photothermal OCT (PTOCT), along with overlaying images of microvascular and tissue morphology. This revealed the utility of PTOCT as part of a powerful multimodality imaging platform for the development of nanomedicines and drug-delivery technologies [62,63] (Fig. 18 10). Kim et al. were able to distinguish gold nanorods and silver nanoplates as OCT contrast agents in the ears of mice that had been intradermally injected [64]. Further improvements in bionanomaterial fabrication to enhance monodispersity and homogeneity improved their scattering resonance intensity, where the two nanomaterials were still detectable in vivo based on their differential scattering characteristics at the respective wavelengths [62].

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FIGURE 18–9 Characterization of pdop-AuNPs and MP-SERS nanoprobes. (A) TEM images of pdop-AuNPs. (B) TEM image of synthesized myoglobin (Mb)-functionalized pdop-AuNP. (C) SEM image of MP-SERS nanoprobes. (D) TEM images of MP-SERS nanoprobes. (E) UV vis spectra of citrate-AuNPs, pdop-AuNPs, Mb-functionalized pdop-AuNPs, MPSERS nanoprobes, and Mb. (F) FEM simulation image of MP-SERS nanoprobe showing a number of hotspots. FEM, finite element method; Mb, Myoglobin; MP-SERS, myoglobin- and polydopamine-engineered surface-enhanced Raman scattering spectroscopy; SEM, scanning electron microscopy; TEM, transmission electron microscopy; UV, ultraviolet. Reproduced with permission from S. Kumar, A. Kumar, G.H. Kim, W.K. Rhim, K.L. Hartman, J.M. Nam, Myoglobin and polydopamine-engineered Raman nanoprobes for detecting, imaging, and monitoring reactive oxygen species in biological samples and living cells, Small 13 (43) (2017) 1701584.

18.3.5 Photoacoustic Imaging PAI harvests the advantages of both optical and acoustic imaging by directing (laser) light pulses into a sample and receiving acoustic information in the form of ultrasound to create images [65]. In PAI, nonionizing laser pulses are delivered into biological tissues (when radio

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FIGURE 18–10 DS- and HH/HVHH-OCT data from nanorod-containing mucus after treatment of isotonic saline. DS, Diffusion-sensitive; HH, horizontal in and horizontal out; HVHH, horizontal in and vertical out; OCT, optical coherence tomography. Reproduced with permission from J. Son, G. Yi, J. Yoo, C. Park, H. Koo, H.S. Choi, Lightresponsive nanomedicine for biophotonic imaging and targeted therapy, Adv. Drug Deliv. Rev. (2018). http://dx.doi. org/10.1002/smll.201701584.

frequency pulses are used, the technology is referred to as thermoacoustic imaging) [66]. Some of the delivered energy will be absorbed and converted into heat, leading to transient thermos elastic expansion and thus wideband (i.e., MHz) ultrasonic emission [67]. The generated ultrasonic waves are detected by ultrasonic transducers and then analyzed to produce images. It is known that optical absorption is closely associated with physiological properties, such as hemoglobin concentration and oxygen saturation. As a result, the magnitude of the

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ultrasonic emission (i.e., photoacoustic signal), which is proportional to the local energy deposition, reveals physiologically specific optical absorption contrast [68]. In this way, 2D or volumetric images of the targeted areas can then be formed. The PAI subsection could fit easily within the optical and acoustic bionanomaterial sections. It fits better in the optical section because the nanomaterials must display to be highly effective PAI contrast agents and must be excellent absorbers of light. Although bionanomaterials have been applied in PAI for many years, recent nanochemistries have led to 100-fold or more improved sensitivities so that PAI can now detect nanomaterial concentrations at picomolar levels [69 71]. Some of these bionanomaterials are innovative and distinctive for PAI; for example, CuS nanoparticles and inorganic graphene analogs such as near-infrared absorbing TiS2 nanosheets. Others such as gold nanomaterials can be optimized for PAI by geometric modification, including spheres, rods, prisms, bipyramids, shells, stars, cages, and vesicles [72]. Some of the latest developments in PAI probes involve smart or activatable nanomaterials that are off until they are turned on by molecules of interest to signal some pathological states. An approach to deep-tissue imaging of ROS in vivo was implemented by semiconductor polymer nanomaterials (SPN). A series of polymeric materials were demonstrated to work such as poly(2,7-(9,9-dioctylfluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole) (PFODBT) and poly[(9,90-dioctyl-2,7-divinylene-fluorenylene)-alt-2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene], among others [73,74]. By this approach, the authors were able to clearly detect and localize the ROS formed in response to acute edema in a mouse model [75] (Fig. 18 11).

FIGURE 18–11 PAI of ROS variation in the tumor of living mice along with drug treatment using the PCBP. (A) Illustration of the mechanism for PAI of ROS in tumor using PCBP. PCBP first accumulates into tumor through the EPR effect and then is activated by ROS to self-assemble and regrow into large nanoparticles, eventually resulting in enhanced photoacoustic signals. (B) Representative PAI maximum-intensity projection images of tumors for BSOpretreated and untreated mice after systemic administration of PCBP (30 µg per mouse) through tail vein. BSO, D,LButhionine-(S,R)-sulphoximine; EPR, Enhanced permeation and retention; PAI, photoacoustic imaging; PCBP, macromolecular probe; ROS, reactive oxygen species. Reproduced with permission from C. Xie, X. Zhen, Y. Lyu, K. Pu, Nanoparticle regrowth enhances photoacoustic signals of semiconducting macromolecular probe for in vivo imaging, Adv. Mater. 29 (44) (2017) 1703693.

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The SPN (PFODBT) for ROS detection was made via a polymeric nanoprecipitation procedure. The PAI signal of the new SPN proved to be several-fold higher than other excellent nanomaterial optical absorbers that are generally used such as single-walled carbon nanotubes (SWCNTs) and gold nanorods based on mass. PAI can be done at sufficient tissue depths and will likely be done at sufficiently low costs to make monitoring of ROS in real time a viable application in clinical medicine [76]. Other nanomaterials have also been developed for detection of enzymatic activity deep within living subjects for pinpointing abnormal or pathological processes [77]. These strategies typically employ a molecule linked to a PAI-active nanomaterial via an enzymatically cleavable peptide. The molecule is often a quencher or a protecting group such that, once cleaved, the PAI signal can be detected [78].

18.4 Acoustic Contrast-Enhancing Bionanomaterials The primary modality applied in the realm of acoustic imaging is ultrasound. Ultrasonic technology has become a major clinical tool for soft-tissue imaging at high penetration depths due to its safety, low cost, and ease of operation [79]. Ultrasonic imaging is a diagnostic imaging tool that uses high-frequency sound waves to create images of structures in biological tissues at high speed with relatively low resolution [80,81]. Nanobubbles have been utilized in acoustic imaging because the compressibility of their gas cores is greater than that of normal tissues [82]. Unfortunately, because contrast interactions with acoustic waves require reflection, acoustic impedance mismatch, refraction, attenuation, or diffraction, nanoscale materials are often poor alternatives to their larger relatives with respect to contrast strength [83]. This poses a challenging technical problem in the field, a tightrope-walking maneuver as a palatable compromise is sought between the smallernanosized regime for favorable in vivo biodistributions and kinetics is balanced by the need for sufficient echogenicity to enhance contrast [84,85]. One of the greatest challenges is to avoid the use of unstable gases, and even gases at all, in nanoscale ultrasonic contrast. For silica and similar glass-based nanomaterials, the contrast is produced by a slightly different mechanism than the bubbles, which display a higher impedance mismatch with the tissues than soft-shelled materials (Fig. 18 12) [86 88]. Smaller nanomaterials are being explored for their echogenicity, and some perhaps will need to be reengineered to achieve sufficient contrast in ultrasound. Surprisingly, a class of carbon nanomaterials known for their contrast and applications in many other modalities (such as optical, photoacoustic, etc.) has also been demonstrated to display ultrasonic contrast in vivo. For example, multiwalled carbon nanotubes, 20 30 nm in diameter and 400 nm in length and thus likely of dimensions able to extravagate from many diseased blood vessels, displayed detectable ultrasonic contrast in living animals such as mice and pigs [90,91]. The strength of the signal observed was at the same level as clinically used sulfur hexafluoride microbubbles, yet the signal did not degrade, as rapidly occurs with microbubbles.

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FIGURE 18–12 (A and E) TEM images of SSN, (B and F) MSN, (C and G) MCF, and (D and H) ELS. Both SSN and MSN were spherical, while MCF and ELS had flatter surfaces. (E H) High-resolution TEM images showing that all particles except SSN were porous. (I) Ultrasound images of ELS showing increased echogenicity of hMSCs. ELS, exosome-like silica nanoparticles; hMSCs, human mesenchymal stem cells; MCF, mesocellular foam silica nanoparticles; MSN, mesoporous silica nanospheres; SSN, Stöber silica nanospheres; TEM, transmission electron microscopy. Reproduced with permission from F. Chen, M. Ma, J. Wang, F. Wang, S.X. Chern, E.R. Zhao, et al., Exosome-like silica nanoparticles: a novel ultrasound contrast agent for stem cell imaging, Nanoscale 9 (1) (2017) 402 411 [89].

18.5 X-Ray Contrast-Enhancing Bionanomaterials As the most commonly used form of radiography, X-ray CT (CT for short hereafter) is among many other forms of tomographic and nontomographic radiography [92]. CT is an imaging technology that can image bionanomaterials that attenuate high-energy radiation [93 95]. CT produces data that can be manipulated to demonstrate various bodily structures based on their ability to absorb an X-ray beam [96]. Although historically the images generated are in the axial or transverse plane perpendicular to the long axis of the body, modern scanners have allowed this volume of data to be reformatted in various planes or even as volumetric (3D) representations of structures. CT has long been a common technique in clinical imaging, used traditionally for bone imaging due to the high contrast between bone and soft tissues [97,98]. As such, denser materials (such as gold over iodine) with higher absorption coefficients make better nanomaterial contrast structures. Contrast can be derived from differences in material atomic number, and for nanomaterial imaging, by far the simplest way to increase

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FIGURE 18–13 CT scans demonstrating migration of gold nanoparticle-labeled T-cells and their whole-body biodistribution. (A) TEM images of spherical AuNPs of increasing size, from 15 to 150 nm. (B) 3D volume rendering CT image of T-cells that accumulated in the lungs 48 h postinjection. Yellow areas represent AuNP-labeled T-cells. (C) Representative 2D CT image of lungs. Arrow indicates gold-labeled cells. (D) Maximum-intensity projection of micro-CT scans 48 h postinjection. Circle demarcates T-cell accumulation in the tumor area. 2D, Two dimensions; 3D, three dimensions; CT, computed tomography; TEM, transmission electron microscopy. Reproduced with permission from R. Meir, R. Popovtzer, Cell tracking using gold nanoparticles and computed tomography imaging, Nanomed. Nanobiotechnol. 10 (2) (2018) e1480.

contrast/signal is thus to increase the atomic number of nanomaterials [99]. In addition, the critical issue in nanomaterial CT contrast agents is a lack of sensitivity. The minimum detectable concentration difference between target and background is on the order of mM, which is estimated to be 5.9 mM for gold [100] (Fig. 18 13). Therefore, the key bottleneck aside from safety/toxicity is the total accumulation of nanomaterials into the target site, making agent delivery perhaps the most important factor driving the viability of CT nanomaterials as clinical diagnostic agents [101].

18.6 Conclusions In recent years, tremendous efforts have been made in applying nanomaterials for in vivo imaging, advancing the course of human disease diagnosis. This chapter has illustrated the common

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imaging modalities and bionanomaterials as contrast agents to enhance their imaging capacities. Although bionanomaterials show many advantages, they also possess lasting limitations such as concerns related to their biosafety. Further optimizations are still required to improve the performance of bionanomaterials in facilitating their eventual clinical translations.

References [1] Y. Gao, Carbon nano-allotrope/magnetic nanoparticle hybrid nanomaterials as T2 contrast agents for magnetic resonance imaging applications, J. Funct. Biomater. 9 (1) (2018) 16. [2] G.M. Nicolle, F. Yerly, D. Imbert, U. Bottger, J.C. Bunzli, A.E. Merbach, Towards binuclear polyaminocarboxylate MRI contrast agents? Spectroscopic and MD study of the peculiar aqueous behavior of the LnIII chelates of OHEC (Ln 5 Eu, Gd, and Tb): implications for relaxivity, Chemistry 9 (22) (2003) 5453 5467. [3] T. Astner, J. Gugler, A. Angerer, S. Wald, S. Putz, N.J. Mauser, et al., Solid-state electron spin lifetime limited by phononic vacuum modes, Nat. Mater. 17 (4) (2018) 313 317. [4] S.C.L. Deoni, S.C.R. Williams, P. Jezzard, J. Suckling, D.G.M. Murphy, D.K. Jones, Standardized structural magnetic resonance imaging in multicentre studies using quantitative T1 and T2 imaging at 1.5 T, Neuroimage 40 (2) (2008) 662 671. [5] X. Jing, Z. Zhi, D. Wang, J. Liu, Y. Shao, L. Meng, Multifunctional nanoflowers for simultaneous multimodal imaging and high-sensitivity chemo-photothermal treatment, Bioconjug. Chem. 29 (2) (2018) 559 570. [6] R. Chen, D. Ling, L. Zhao, S. Wang, Y. Liu, R. Bai, et al., Parallel comparative studies on mouse toxicity of oxide nanoparticle- and gadolinium-based T1 MRI contrast agents, ACS Nano 9 (12) (2015) 12425 12435. [7] H.B. Na, J.H. Lee, K. An, Y.I. Park, M. Park, I.S. Lee, et al., Development of a T1 contrast agent for magnetic resonance imaging using MnO nanoparticles, Angew. Chem. Int. Ed. Engl. 46 (28) (2007) 5397 5401. [8] J. Li, C. Wu, P. Hou, M. Zhang, K. Xu, One-pot preparation of hydrophilic manganese oxide nanoparticles as T1 nano-contrast agent for molecular magnetic resonance imaging of renal carcinoma in vitro and in vivo, Biosens. Bioelectron. 102 (2018) 1 8. [9] Q. Wang, A. Xiao, Y. Liu, Q. Zou, Q. Zhou, H. Wang, et al., One-step preparation of nano-in-micro poly (vinyl alcohol) embolic microspheres and used for dual-modal T1/T2-weighted magnetic resonance imaging, Nanomedicine 14 (8) (2018) 2551 2561. [10] C. Burtea, S. Laurent, L. Vander Elst, R.N. Muller, Contrast agents: magnetic resonance, Handb. Exp. Pharmacol. 185 Pt 1 (2008) 135 165. [11] E. Li, Y. Yang, G. Hao, X. Yi, S. Zhang, Y. Pan, et al., Multifunctional magnetic mesoporous silica nanoagents for in vivo enzyme-responsive drug delivery and MR imaging, Nanotheranostics 2 (3) (2018) 233 242. [12] Q. Peng, D. Huo, H. Li, B. Zhang, Y. Li, A. Liang, et al., ROS-independent toxicity of Fe3O4 nanoparticles to yeast cells: Involvement of mitochondrial dysfunction, Chem. Biol. Interact. 287 (2018) 20 26. [13] J. Gallo, N. Kamaly, I. Lavdas, E. Stevens, Q.D. Nguyen, M. Wylezinska-Arridge, et al., CXCR4-targeted and MMP-responsive iron oxide nanoparticles for enhanced magnetic resonance imaging, Angew. Chem. Int. Ed. Engl. 53 (36) (2014) 9550 9554. [14] L.L. Feng, C.Q. Wang, C.X. Li, S.L. Gai, F. He, R.M. Li, et al., Multifunctional theranostic nanoplatform based on Fe-mTa(2)O(5)@CuS-ZnPc/PCM for bimodal imaging and synergistically enhanced phototherapy, Inorg. Chem. 57 (9) (2018) 4864 4876.

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[15] A. Ahmad, H. Bae, I. Rhee, Highly stable silica-coated manganese ferrite nanoparticles as high-efficacy T-2 contrast agents for magnetic resonance imaging, AIP Adv. 8 (5) (2018) 9. [16] T. Kluth, Mathematical models for magnetic particle imaging, Inverse Probl. 34 (8) (2018) 27. [17] H. Rogge, M. Erbe, T.M. Buzug, K. Ludtke-Buzug, Simulation of the magnetization dynamics of diluted ferrofluids in medical applications, Biomed. Tech. (Berl) 58 (6) (2013) 601 609. [18] Y. Du, P.T. Lai, C.H. Leung, P.W. Pong, Design of superparamagnetic nanoparticles for magnetic particle imaging (MPI), Int. J. Mol. Sci. 14 (9) (2013) 18682 18710. [19] H. Arami, R.M. Ferguson, A.P. Khandhar, K.M. Krishnan, Size-dependent ferrohydrodynamic relaxometry of magnetic particle imaging tracers in different environments, Med. Phys. 40 (7) (2013) 071904. [20] A.P. Khandhar, P. Keselman, S.J. Kemp, R.M. Ferguson, P.W. Goodwill, S.M. Conolly, et al., Evaluation of PEG-coated iron oxide nanoparticles as blood pool tracers for preclinical magnetic particle imaging, Nanoscale 9 (3) (2017) 1299 1306. [21] H. Kratz, M. Taupitz, A.A. de Schellenberger, O. Kosch, D. Eberbeck, S. Wagner, et al., Novel magnetic multicore nanoparticles designed for MPI and other biomedical applications: from synthesis to first in vivo studies, PLoS One 13 (1) (2018) 22. [22] E.Y. Yu, P. Chandrasekharan, R. Berzon, Z.W. Tay, X.Y. Zhou, A.P. Khandhar, et al., Magnetic particle imaging for highly sensitive, quantitative, and safe in vivo gut bleed detection in a murine model, ACS Nano 11 (12) (2017) 12067 12076. [23] T.H. Shin, Y. Choi, S. Kim, J. Cheon, Recent advances in magnetic nanoparticle-based multi-modal imaging, Chem. Soc. Rev. 44 (14) (2015) 4501 4516. [24] M. Mehrmohammadi, K.Y. Yoon, M. Qu, K.P. Johnston, S.Y. Emelianov, Enhanced pulsed magnetomotive ultrasound imaging using superparamagnetic nanoclusters, Nanotechnology 22 (4) (2011) 045502. [25] K.Y. Yoon, M. Mehrmohammadi, A. Borwankar, S.Y. Emelianov, K.P. Johnston, Synthesis of iron oxide nanoclusters with enhanced magnetization and their applications in pulsed magneto-motive ultrasound imaging, Nano 10 (5) (2015) 9. [26] B. Arnal, S.J. Yoon, J.W. Li, X.H. Gao, M. O’Donnell, Magneto-optical nanoparticles for cyclic magnetomotive photoacoustic imaging, Physica C 548 (2018) 90 92. [27] M. Mehrmohammadi, T.H. Shin, M. Qu, P. Kruizinga, R.L. Truby, J.H. Lee, et al., In vivo pulsed magneto-motive ultrasound imaging using high-performance magnetoactive contrast nanoagents, Nanoscale 5 (22) (2013) 11179 11186. [28] R. Cheng, D. Li, H. Zhou, C. Wang, A. Yin, S. Jiang, et al., Electroluminescence and photocurrent generation from atomically sharp WSe2/MoS2 heterojunction p n diodes, Nano Lett. 14 (10) (2014) 5590 5597. [29] M. Yang, Y. Liang, Q. Gui, B. Zhao, D. Jin, M. Lin, et al., Multifunctional luminescent nanomaterials from NaLa(MoO4)2:Eu(3 1 )/Tb(3 1 ) with tunable decay lifetimes, emission colors, and enhanced cell viability, Sci. Rep. 5 (2015) 11844. [30] J. Park, A. Estrada, K. Sharp, K. Sang, J.A. Schwartz, D.K. Smith, et al., Two-photon-induced photoluminescence imaging of tumors using near-infrared excited gold nanoshells, Opt. Express 16 (3) (2008) 1590 1599. [31] Z. Deng, L. Tong, M. Flores, S. Lin, J.X. Cheng, H. Yan, et al., High-quality manganese-doped zinc sulfide quantum rods with tunable dual-color and multiphoton emissions, J. Am. Chem. Soc. 133 (14) (2011) 5389 5396. [32] M.K. Tsang, G. Bai, J. Hao, Stimuli responsive upconversion luminescence nanomaterials and films for various applications, Chem. Soc. Rev. 44 (6) (2015) 1585 1607. [33] L. Su, X. Zhang, Y. Zhang, A.L. Rogach, Recent progress in quantum dot based white light-emitting devices, Top. Curr. Chem. (Cham) 374 (4) (2016) 42.

418

THERANOSTIC BIONANOMATERIALS

[34] C. Wang, X. Li, F. Zhang, Bioapplications and biotechnologies of upconversion nanoparticle-based nanosensors, Analyst 141 (12) (2016) 3601 3620. [35] L. Gan, X. Li, X. Cai, K. Liu, W. Li, S.J. Su, D-A-D-type orange-light emitting thermally activated delayed fluorescence (TADF) materials based on a fluorenone unit: simulation, photoluminescence and electroluminescence studies, Beilstein J. Org. Chem. 14 (2018) 672 681. [36] I.C. Kong, K.S. Ko, M.H. Lee, J.H. Lee, Y.H. Han, Ecotoxicity evaluation of Cu- and Fe-CNT complexes based on the activity of bacterial bioluminescence and seed germination, J. Environ. Sci. (China) 67 (2018) 198 205. [37] M. Sentic, S. Arbault, L. Bouffier, D. Manojlovic, A. Kuhn, N. Sojic, 3D electrogenerated chemiluminescence: from surface-confined reactions to bulk emission, Chem. Sci. 6 (8) (2015) 4433 4437. [38] L. Zhu, M.T. Trinh, L. Yin, Z. Zhang, Sequential oligodiacetylene formation for progressive luminescent color conversion via co-micellar strategy, Chem. Sci. 7 (3) (2016) 2058 2065. [39] A.A. Tukhbatullin, G.L. Sharipov, A.A. Galina, Mechanoluminescence of Ce/Tb inorganic salts in methane-acetylene mixtures with inert gases, Luminescence 33 (7) (2018) 1180 1184. [40] S.M. King, S. Claire, R.I. Teixeira, A.N. Dosumu, A.J. Carrod, H. Dehghani, et al., Iridium nanoparticles for multichannel luminescence lifetime imaging, mapping localization in live cancer cells, J. Am. Chem. Soc. (2018). [41] T. Ai, W.T. Shang, H. Yan, C.T. Zeng, K. Wang, Y. Gao, et al., Near infrared-emitting persistent luminescent nanoparticles for Hepatocellular Carcinoma imaging and luminescence-guided surgery, Biomaterials 167 (2018) 216 225. [42] T. Ai, W. Shang, H. Yan, C. Zeng, K. Wang, Y. Gao, et al., Near infrared-emitting persistent luminescent nanoparticles for Hepatocellular Carcinoma imaging and luminescence-guided surgery, Biomaterials 167 (2018) 216 225. [43] M. Kownacki, S.M. Langenegger, S.X. Liu, R. Haner, Integrating DNA photonic wires into lightharvesting supramolecular polymers, Angew. Chem. Int. Ed. Engl. 58 (3) (2019) 751 755. [44] S. Takahashi, A. Yoshida, H. Oikawa, Hypothesis: structural heterogeneity of the unfolded proteins originating from the coupling of the local clusters and the long-range distance distribution, Biophys. Rev. 10 (2) (2018) 363 373. [45] E. Sobakinskaya, M. Schmidt Am Busch, T. Renger, Theory of FRET “Spectroscopic Ruler” for short distances: application to polyproline, J. Phys. Chem. B 122 (1) (2018) 54 67. [46] M. Yang, S. Peng, R. Sun, J. Lin, N. Wang, C. Chen, The conformational dynamics of Cas9 governing DNA cleavage are revealed by single-molecule FRET, Cell Rep. 22 (2) (2018) 372 382. [47] J. Yoo, J.M. Louis, I.V. Gopich, H.S. Chung, Three-color single-molecule FRET and fluorescence lifetime analysis of fast protein folding, J. Phys. Chem. B 122 (49) (2018) 11702 11720. [48] X. Gao, B. Zhang, Q. Zhang, Y. Tang, X. Liu, J. Li, The influence of combination mode on the structure and properties of graphene quantum dot-porphyrin composites, Colloids Surf. B Biointerfaces 172 (2018) 207 212. [49] N.S. Annamdevula, R. Sweat, J.R. Griswold, K. Trinh, C. Hoffman, S. West, et al., Spectral imaging of FRET-based sensors reveals sustained cAMP gradients in three spatial dimensions, Cytometry A 93 (10) (2018) 1029 1038. [50] R. Petrovics, B. Soveges, A. Egyed, G. Knorr, A. Kormos, T. Imre, et al., A rapid and concise setup for the fast screening of FRET pairs using bioorthogonalized fluorescent dyes, Org. Biomol. Chem. 16 (16) (2018) 2997 3005. [51] R. Liu, L. Zhang, Y. Chen, Z. Huang, Y. Huang, S. Zhao, Design of a new near-infrared ratiometric fluorescent nanoprobe for real-time imaging of superoxide anions and hydroxyl radicals in live cells and in situ tracing of the inflammation process in vivo, Anal. Chem. 90 (7) (2018) 4452 4460.

Chapter 18 • Bionanomaterials as Imaging Contrast Agents

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[52] S. Yerolatsitis, F. Yu, S. McAughtrie, M.G. Tanner, H. Fleming, J.M. Stone, et al., Ultra-low background Raman sensing using a negative-curvature fibre and no distal optics, J. Biophotonics 12 (2018) e201800239. [53] Y. Fuentes-Edfuf, M. Garcia-Lechuga, D. Puerto, C. Florian, A. Garcia-Leis, S. Sanchez-Cortes, et al., Coherent scatter-controlled phase-change grating structures in silicon using femtosecond laser pulses, Sci. Rep. 7 (1) (2017) 4594. [54] E. Bae, H. Kim, B. Rajwa, J.G. Thomas, J.P. Robinson, Current status and future prospects of using advanced computer-based methods to study bacterial colonial morphology, Expert Rev. Anti-Infect. Ther. 14 (2) (2016) 207 218. [55] F.C. Clarke, M.J. Jamieson, D.A. Clark, S.V. Hammond, R.D. Jee, A.C. Moffat, Chemical image fusion. The synergy of FT-NIR and Raman mapping microscopy to enable a more complete visualization of pharmaceutical formulations, Anal. Chem. 73 (10) (2001) 2213 2220. [56] C.G. Artur, T. Womack, F.S. Zhao, J.L. Eriksen, D. Mayerich, W.C. Shih, Plasmonic nanoparticle-based expansion microscopy with surface-enhanced Raman and dark-field spectroscopic imaging, Biomed. Opt. Express 9 (2) (2018) 603 615. [57] S. Kumar, A. Kumar, G.H. Kim, W.K. Rhim, K.L. Hartman, J.M. Nam, Myoglobin and polydopamineengineered Raman nanoprobes for detecting, imaging, and monitoring reactive oxygen species in biological samples and living cells, Small 13 (43) (2017) 1701584. [58] K.H. Chan, D. Fried, Multispectral cross-polarization reflectance measurements suggest high contrast of demineralization on tooth surfaces at wavelengths beyond 1300 nm due to reduced light scattering in sound enamel, J. Biomed. Opt. 23 (6) (2018) 1 4. [59] B.R. Smith, S.S. Gambhir, Nanomaterials for in vivo imaging, Chem. Rev. 117 (3) (2017) 901 986. [60] R.Z. Seeni, X.J. Yu, H. Chang, P. Chen, L.B. Liu, C.J. Xu, Iron oxide nanoparticle-powered micro-optical coherence tomography for in situ imaging the penetration and swelling of polymeric microneedles in the skin, ACS Appl. Mater. Interfaces 9 (24) (2017) 20340 20347. [61] F.A. Jaffer, P. Libby, R. Weissleder, Optical and multimodality molecular imaging: insights into atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 29 (7) (2009) 1017 1024. [62] J. Son, G. Yi, J. Yoo, C. Park, H. Koo, H.S. Choi, Light-responsive nanomedicine for biophotonic imaging and targeted therapy, Adv. Drug Deliv. Rev. (2018). http://dx.doi.org/10.1002/smll.201701584. [63] J.M. Tucker-Schwartz, K.R. Beavers, W.W. Sit, A.T. Shah, C.L. Duvall, M.C. Skala, In vivo imaging of nanoparticle delivery and tumor microvasculature with multimodal optical coherence tomography, Biomed. Opt. Express 5 (6) (2014) 1731 1743. [64] D. Sen, E.D. SoRelle, O. Liba, R. Dalal, Y.M. Paulus, T.W. Kim, et al., High-resolution contrast-enhanced optical coherence tomography in mice retinae, J. Biomed. Opt. 21 (6) (2016) 66002. [65] R.G. Kolkman, M.J. Mulder, C.P. Glade, W. Steenbergen, T.G. van Leeuwen, Photoacoustic imaging of port-wine stains, Lasers Surg. Med. 40 (3) (2008) 178 182. [66] P.G. Sang, J. Heo, H.J. Park, H.W. Baac, Photoacoustic energy sensor for nanosecond optical pulse measurement, Sensors (Basel) 18 (11) (2018). [67] T. Petrosyan, M. Theodorou, J. Bamber, M. Frenz, M. Jaeger, Rapid scanning wide-field clutter elimination in epi-optoacoustic imaging using comb LOVIT, Photoacoustics 10 (2018) 20 30. [68] M. Li, Y. Tang, J. Yao, Photoacoustic tomography of blood oxygenation: a mini review, Photoacoustics 10 (2018) 65 73. [69] P. Zhao, Z. Jin, Q. Chen, T. Yang, D. Chen, J. Meng, et al., Local generation of hydrogen for enhanced photothermal therapy, Nat. Commun. 9 (1) (2018) 4241. [70] B. Wu, S.T. Lu, H. Yu, R.F. Liao, H. Li, B.V. Lucie Zafitatsimo, et al., Gadolinium-chelate functionalized bismuth nanotheranostic agent for in vivo MRI/CT/PAI imaging-guided photothermal cancer therapy, Biomaterials 159 (2018) 37 47.

420

THERANOSTIC BIONANOMATERIALS

[71] L. Zhou, Y. Jing, Y. Liu, Z. Liu, D. Gao, H. Chen, et al., Mesoporous carbon nanospheres as a multifunctional carrier for cancer theranostics, Theranostics 8 (3) (2018) 663 675. [72] M. Mendes, J.J. Sousa, A. Pais, C. Vitorino, Targeted theranostic nanoparticles for brain tumor treatment, Pharmaceutics 10 (4) (2018). [73] J. Qi, Y. Fang, R.T.K. Kwok, X. Zhang, X. Hu, J.W.Y. Lam, et al., Highly stable organic small molecular nanoparticles as an advanced and biocompatible phototheranostic agent of tumor in living mice, ACS Nano 11 (7) (2017) 7177 7188. [74] P. Manivasagan, S. Bharathiraja, N.Q. Bui, I.G. Lim, J. Oh, Paclitaxel-loaded chitosan oligosaccharidestabilized gold nanoparticles as novel agents for drug delivery and photoacoustic imaging of cancer cells, Int. J. Pharm. 511 (1) (2016) 367 379. [75] C. Xie, X. Zhen, Y. Lyu, K. Pu, Nanoparticle regrowth enhances photoacoustic signals of semiconducting macromolecular probe for in vivo imaging, Adv. Mater. 29 (44) (2017) 1703693. [76] I.S. Al Rowaihi, A. Paillier, S. Rasul, R. Karan, S.W. Grotzinger, K. Takanabe, et al., Poly(3-hydroxybutyrate) production in an integrated electromicrobial setup: investigation under stress-inducing conditions, PLoS One 13 (4) (2018) e0196079. [77] T.D. Berry, T.R. Filley, R.A. Blanchette, Oxidative enzymatic response of white-rot fungi to single-walled carbon nanotubes, Environ. Pollut. 193 (2014) 197 204. [78] S. Onoe, Development of molecular probes for spatio-temporal analysis of in vivo tumor with photoacoustic imaging, Yakugaku Zasshi 136 (3) (2016) 491 498. [79] H. Wen, R.S. Balaban, Ultrasonic imaging of the electroacoustic effect in macromolecular gels, Ultrason. Imaging 20 (4) (1998) 288 297. [80] D.H. Turnbull, S.R. Wilson, A.L. Hine, F.S. Foster, Ultrasonic characterization of selected renal tissues, Ultrasound Med. Biol. 15 (3) (1989) 241 253. [81] E. Zhang, P. Beard, Broadband ultrasound field mapping system using a wavelength tuned, optically scanned focused laser beam to address a Fabry Perot polymer film sensor, IEEE Trans. Ultrason. Ferroelectr. Freq. Control 53 (7) (2006) 1330 1338. [82] M.H. Chan, Y.T. Pan, Y.C. Chan, M. Hsiao, C.H. Chen, L. Sun, et al., Nanobubble-embedded inorganic 808 nm excited upconversion nanocomposites for tumor multiple imaging and treatment, Chem. Sci. 9 (12) (2018) 3141 3151. [83] M. Li, H. Luo, W. Zhang, K. He, Y. Chen, J. Liu, et al., Phase-shift, targeted nanoparticles for ultrasound molecular imaging by low intensity focused ultrasound irradiation, Int. J. Nanomed. 13 (2018) 3907 3920. [84] K. Minaga, M. Takenaka, S. Omoto, T. Miyata, K. Kamata, K. Yamao, et al., A case of successful transluminal drainage of walled-off necrosis under contrast-enhanced harmonic endoscopic ultrasonography guidance, J. Med. Ultrason. 45 (1) (2018) 161 165. [85] S. Houvenagel, L. Moine, G. Picheth, C. Dejean, A. Brulet, A. Chenneviere, et al., Comb-like fluorophilic-lipophilic-hydrophilic polymers for nanocapsules as ultrasound contrast agents, Biomacromolecules 19 (8) (2018) 3244 3256. [86] H. Chen, D. Zheng, J. Liu, Y. Kuang, Q. Li, M. Zhang, et al., pH-Sensitive drug delivery system based on modified dextrin coated mesoporous silica nanoparticles, Int. J. Biol. Macromol. 85 (2016) 596 603. [87] K. Krishna Priya, M. Ramesh, M. Saravanan, N. Ponpandian, Ecological risk assessment of silicon dioxide nanoparticles in a freshwater fish Labeo rohita: hematology, ionoregulation and gill Na(1)/K(1) ATPase activity, Ecotoxicol. Environ. Saf. 120 (2015) 295 302. [88] C.H. Tsai, J.L. Vivero-Escoto, I.I. Slowing, I.J. Fang, B.G. Trewyn, V.S. Lin, Surfactant-assisted controlled release of hydrophobic drugs using anionic surfactant templated mesoporous silica nanoparticles, Biomaterials 32 (26) (2011) 6234 6244.

Chapter 18 • Bionanomaterials as Imaging Contrast Agents

421

[89] F. Chen, M. Ma, J. Wang, F. Wang, S.X. Chern, E.R. Zhao, et al., Exosome-like silica nanoparticles: a novel ultrasound contrast agent for stem cell imaging, Nanoscale 9 (1) (2017) 402 411. [90] H. Kafa, J.T. Wang, N. Rubio, R. Klippstein, P.M. Costa, H.A. Hassan, et al., Translocation of LRP1 targeted carbon nanotubes of different diameters across the blood brain barrier in vitro and in vivo, J. Control. Release 225 (2016) 217 229. [91] H. Kafa, J.T. Wang, N. Rubio, K. Venner, G. Anderson, E. Pach, et al., The interaction of carbon nanotubes with an in vitro blood brain barrier model and mouse brain in vivo, Biomaterials 53 (2015) 437 452. [92] D.G. Tobert, H.V. Le, J.A. Blucher, M.B. Harris, A.J. Schoenfeld, The clinical implications of adding CT angiography in the evaluation of cervical spine fractures: a propensity-matched analysis, J. Bone Joint Surg. Am. 100 (17) (2018) 1490 1495. [93] E. Li, X. Cheng, Y. Deng, J. Zhu, X. Xu, P.E. Saw, et al., Fabrication of PEGylated Fe@Bi2S3 nanocomposites for dual-mode imaging and synergistic thermoradiotherapy, Biomater. Sci. 6 (7) (2018) 1892 1898. [94] N. Fulton, P. Rajiah, Abdominal applications of a novel detector-based spectral CT, Curr. Probl. Diagn. Radiol. 47 (2) (2018) 110 118. [95] A. Ramon, A. Bohm-Sigrand, P. Pottecher, P. Richette, J.F. Maillefert, H. Devilliers, et al., Role of dualenergy CT in the diagnosis and follow-up of gout: systematic analysis of the literature, Clin. Rheumatol. 37 (3) (2018) 587 595. [96] G.J. Gang, W. Zbijewski, M. Mahesh, G. Thawait, N. Packard, J. Yorkston, et al., Image quality and dose for a multisource cone-beam CT extremity scanner, Med. Phys. 45 (1) (2018) 144 155. [97] K. Tallroth, Plain CT of the degenerative lumbar spine, Eur. J. Radiol. 27 (3) (1998) 206 213. [98] L. Shi, W. Liu, L. Yin, S. Feng, S. Xu, Z.Y. Zhang, Surgical guide assistant mandibular distraction osteogenesis and sagittal split osteotomy in the treatment of hemifacial microsomia, J. Craniofac. Surg. 26 (2) (2015) 498 500. [99] X. Zhu, W. Cao, B. Chang, L. Zhang, P. Qiao, X. Li, et al., Polyacrylate/nanosilica causes pleural and pericardial effusion, and pulmonary fibrosis and granuloma in rats similar to those observed in exposed workers, Int. J. Nanomed. 11 (2016) 1593 1605. [100] R. Meir, R. Popovtzer, Cell tracking using gold nanoparticles and computed tomography imaging, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 10 (2) (2018) e1480. [101] M. Obenaus, C. Leitao, M. Leisegang, X. Chen, I. Gavvovidis, P. van der Bruggen, et al., Identification of human T-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice, Nat. Biotechnol. 33 (4) (2015) 402 407.

19 Nucleotide Aptamers as Theranostic Biomaterials Yuezhou Zhang1, Dhayakumar Rajan Prakash1, Hongbo Zhang1,2 1

D E P A R T M E N T O F P H A RM A CE U T I C A L S CI E N C E LA BO RA T O RY , Å BO AK AD E MI UN IVERSITY, TURKU, FINLAND 2 TURKU CENTER FOR BIOTECHNOLOGY, UNIVERSITY OF TURKU A ND ÅBO A KADE MI UNIVERS ITY, TURK U, FINLAN D

19.1 Introduction Nucleotide aptamers are short (20
80 mer), single-stranded (ss) oligonucleotides (DNA or RNA). The word “aptamer” was coined by Eligton and Szotw more than two decades ago [1], and is derived from the Latin “aptus” (meaning “to fit”) and the Greek “meros” (meaning “part or region”). The identification of aptamers is termed systematic evolution of ligands by exponential enrichment (SELEX), also known as in vitro selection or evolution and was first introduced in 1990 [2] and commemorated its quarter century since discovery in a special issue of the Journal of Molecular Evolution published in 2015 [3]. Fig. 19
1 presents the detailed procedure of SELEX [4]. SELEX is the most common terminology to describe this procedure, but selected and amplified binding site and cyclic amplification and selection of targets [5] were also used depending on the researchers’ preference. The process begins with the synthesis oligonucleotide library consisting of a random sequence of generated sequences with fixed length of n, therefore the number of possible sequences is 4n. The library pool usually contains approximately 1014
1016 oligonucleotide strands which are incubated with the target of interest and washed to identify the tightest-binding sequences [6]. Using this technique, aptamers with high affinity and specificity to the targeting molecules can be isolated from large pools of randomized ssDNA or RNA in a highly iterative way that oligonucleotides which bind to a target are eluted and amplified by polymerase chain reaction while the unbound ones are washed away. As a result, highly robust and specific aptamers can be obtained. Hasegawa et al. reviewed the methods applied to improve aptamer binding affinity, such as sequence optimization, structure stabilization, introduction of hydrophobic portions into aptamers, and binding motif conjugation [7]. Modification of the traditional SELEX process has led to cell-SELEX technology [8] which uses whole living cells as targets to generate cell-specific aptamers with a high likelihood of successful application in in vivo assays.

Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00019-5 © 2019 Elsevier Inc. All rights reserved.

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FIGURE 19–1 Schematic representation of the SELEX procedure to produced nucleotide aptamers for specific target biomolecules. SELEX, Systematic evolution of ligands by exponential enrichment. Reproduced with permission from H. Jo, C. Ban, Aptamer
nanoparticle complexes as powerful diagnostic and therapeutic tools, Exp. Mol. Med. 48 (5) (2016) e230. © 2015 Springer Nature.

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As long as the desired nucleotide sequence of the aptamer is determined, stepwise synthesis using an oligonucleotide synthesizer can be implemented [9], a profound step toward clinic application to reduce their production costs compared with other therapeutics. Aptamers are also called “chemical antibodies,” but they provide advantages over classical ones. The production of polypeptidic antibodies requires a large quantity of mammalian cell cultures and lengthy purification steps under strict good manufacturing practice conditions. As a result, the cost of antibody production skyrocketed and prevented wider usage. Given the smaller size, the internalization of aptamer into tissues and target arrival are more efficient than antibodies, especially in the case of solid tumors which account for more than 85% of human cancers [10]. So far, only a few monoclonal antibodies—trastuzumab, cetuximab, panitumumab, bevacizumab, catumaxomab, ipilimumab, and denosumab [11,12]— have been approved by US Food and Drug Administration for cancer therapy related to solid tumors. Two aptamers—AS1411 targeting nucleolin [13] and NOX-A12 targeting C-X-C chemokine ligand 12 (CXCL12) [14]—are currently undergoing clinical trials for treatment of cancer patients, and many more are under study. Aptamers often show low toxicity and lack immunogenicity over conventional antibodies, and therefore can target immune regulatory proteins. In addition, aptamers are thermodynamically stable, which favors the circulation of aptamers in blood. In addition, the stability improvement and multifunctionality of aptamers can be achieved by chemical modification [15]. It is generally believed than fulfilling the chemical modification of aptamers is easier if similar functions are assigned to the antibody [16]. Often the modification of aptamers starts with pretreatment of the aptamer building block nucleotide at a different numbering position. For instance, 50 -biotin is introduced to facilitate the purification, 6-carboxyfluorescein for detection, 20 -O-methyl RNA bases, 20 -fluoro, 20 -thiol, 20 -hydroxymethyl, or 20 -azido for stability enhancement during in vitro and in vivo usage [17]. A comprehensive pros and cons of nucleic acid aptamers versus antibodies were reviewed by Zhou et al. [18]. In this chapter, we mainly discuss the nucleotide aptamer structural composition, therapeutic application, and development prospects as biomaterials.

19.2 The Structure of Aptamers and Complexes 19.2.1 Primary Aptamer Structures When two regions of the same oligonucleotide strand are complementary to each other through Watson
Crick base pairs hairpin (stem)-loops take place. Two hairpin loops form kissing complexes when the unpaired nucleotides in one loop base pair with the unpaired nucleotides in another. G-quadruplex structures, arising in guanine-rich sequences, are helical structures containing quandine tetrads associated through H-bonds. The pseudoknot is an RNA secondary structure containing two interacting stem-loops, as such the loop of one stem-loop is the stem of the other. Driven by intramolecular interactions such as π
π stacking, hydrogen bond (H-bond), van der Waals force, and ionic interaction which are inherited from the nucleotide building

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FIGURE 19–2 Fundamental structure of aptamer: (A) G-quartet. (B) Aptamer loop. (C) Pseudoknot. (A) Reprint with permission from J.A. Capra, K. Paeschke, M. Singh, V.A. Zakian, G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae, PLoS Comput. Biol. 6 (7) (2010) e1000861. © 2010, PLOS Computational Biology, open access. (B) Adapted from J. Da Costa, Catalytic Potential and Ligand Binding Properties of the Malachite Green RNA Aptamer, University of Waterloo, 2008. (C) Reprint with permission from Q. Zhang, R. Landgraf, Selecting molecular recognition. What can existing aptamers tell us about their inherent recognition capabilities and modes of interaction?, Pharmaceuticals 5 (5) (2012) 493
513. © 2012 MDPI, open access.

blocks of aptamers, nucleotide aptamers can form many three-dimensional (3D) structures, such as G-quartet [19] in Fig. 19
2A, bulge and hairpin loop [20] in Fig. 19
2B, and pseudoknot [21] in Fig. 19
2C. Most aptamers bind to proteins with equilibrium constant (Kd) in the range of 1 picomolar to 1 nanomolar [22]; such exceptional molecular recognition contributes to both shape-complementary [23] and noncovalent binding with the target binding domain [24]. The aptamers’ 3D binding pockets and clefts for the specific recognition and tight binding favor the attachment of reporter molecules and surface-acceptors a in specific manner.

19.2.2 Cocrystal Structures of Aptamers With Ligands As the structural features of stranded oligonucleotides are of major importance to their biological function, there is much interest in structure identification, either in the ligand-free (“apo”) state or in complex with various ligands, such as proteins, metabolites. The determination of cocrystal structures of aptamers with their target molecules helps in understanding its super high binding affinity and specificity from a molecule interaction perspective. In the case of small organic compounds, the complex structures show that aptamers form a cage that entraps ligands. For instance, RNA aptamers bind to aminoglycoside antibiotics tobramycin with Kds in the μM range which was refined to create a high affinity aptamer with Kds in the nM range [25]. Neomycin is recognized by selected RNA aptamers with high specificity

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FIGURE 19–3 Costructure of aptamers with (A) small (PDB code: 1NTA) and (B) large (PDB code: 5EW1) ligands. The ligand streptomycin and thrombin are green and the backbones of aptamer are brown. The figures were represented by PyMOL.

(.100-fold higher affinity than for reference paromomycin) [26]. A 2.9 Å crystal structure of streptomycin bound to small 40-mer RNA has defined the intermolecular contact as such a streptomycin streptose ring was trapped by stacked arrays of bases from both loops at the elbow of the L-shaped RNA aptamer (Fig. 19
3A) and specificity is defined by direct H-bonds between all streptose hydroxyl groups and base edges [27]. In structures of aptamers in complex with proteins, the aptamers occupy the nucleic acid binding site of protein, which is subject to H-bonds, ionic interaction, and π
π stacking. Long et al. solved a crystal structure of an RNA aptamer bound to the basic region on a thrombin called exosite-II for heparin; this recognition was significantly addressed by the planar stacking of adenine bases at the core of the aptamer tertiary fold with arginine side chains of proteins (PDB code: 3DD2) [28]. The complex of two DNA aptamers, HD1 and HD22, simultaneously bound to human thrombin was also presented (Fig. 19
3B), in which thrombin was sandwiched by HD1 aptamers at exosite I and HD22 adhered to exosite II [29]. The aptamer RNA-2 adopts remarkably differently when free and when in a complex with Bacillus ribosomal protein S8 (PDB code: 2LUN), which reflected the plasticity of RNA secondary structure [30] and implied the occurrence of induced fit [31] when they interacted [32].

19.2.3 Aptamer Structure Prediction Beside the massive crystallographic work for studying aptamer-target complexes, an attempt to predict the structure of oligonucleotides including aptamers has led to a few computational applications in use [28]. These include OligoCalc for intermolecular selfcomplementarity estimation and intramolecular hairpin loop formation [33], RNAstructure

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package for RNA and DNA secondary structure prediction and analysis [34], 3D-DART webserver to specifically model the 3D structure of DNA molecules in their native form or in complex with a ligand [35], APTANI computationally select aptamers through sequencestructure motif analysis of high-throughput (HT)-SELEX data [36], and HADDOCK portal for protein-nucleic acid docking [37]. Jeddi et al. presented the first approach to predict the 3D structures of 24 ssDNA hairpins precisely, exemplified by a low root-mean-square deviation (RMSD) value of 1.87 Å between predicted structure and corresponding experimental structures downloaded from the PDB (PDB ID: 1PQT) [38]. The high degree of linear correlation (R2 5 0.9789) between the free energy of the experimentally determined binding affinity of thrombin binding aptamer for potassium and the computationally estimated observation demonstrated the validity of in silico tools [39].

19.3 Applications of Aptamers Similar to classical glycoprotein antibodies, aptamers find ever-increasing applications in the biomedical field, serving as medications, diagnostics, analytics, bio-imaging, and aptasensors. A milestone has been the successful commercialization of the first aptamer-based diagnostic kit for the detection of mycotoxins in grains by NeoVentures Biotechnology Inc.

19.3.1 Therapeutical Nucleotide Aptamers 19.3.1.1 Aptamers for Cancer Therapies Given aptamers’ greater precision but fewer side effects, their targeted cancer therapies and potential have been highlighted in cancer research. In contrast to “traditional” cytotoxic chemotherapies which kill dividing cells by interfering with cell division, therapeutic aptamers are aimed at interfering with specific molecules necessary for diseased tissue growth while keeping others intact. AS1411 AS1411 is a guanine-rich (G-rich) quadruplex-forming 26-mer DNA aptamer that binds specifically to nucleolin, a protein overexpressed in the plasma membrane of many types of cancer but absent from the surface/cytoplasm of most normal cells [40]. AS1411, developed by British company Antisoma, is the first-in-class anticancer oligonucleotide currently in phase II clinical trials. In addition, chemical modification of 5-(N-benzyl-carboxyamide)-20 -deoxyuridine (5-BzdU) in the AS1411-aptamer was also under investigation to suppress hepatocellular carcinoma cell growth by increasing aptamer binding affinity [41]. It has been suggested that AS1411 functions as a cell antiproliferative reagent in a nucleolin-dependent manner. ReyesReyes et al. disclosed that AS1411 was taken up in cancer cells via macropinocytosis and the internalization of AS1411 stimulated additional macropinocytosis, therefore mediating further uptake of molecules from the extracellar media [42]. Furthermore, it was found that activation of epidermal growth factor receptor (EGFR) and Rac1 was crucial for AS1411 activity in cancer

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cells (U145, MDA-MB-468, A549, LNCaP) and their inhibition significantly reduced AS1411induced macropinocytosis and its antiproliferative activity. Cheng et al. also reported that AS1411 induced cell apoptosis, cycle arrest, and inhibited cell viability by upregulation of p53 and downregulation of Bcl-2 and Akt1 in human glioma cells via Nucleolin [43]. NOX-A12 NOX-A12, a 45-mer L-RNA aptamer (Spiegelmer) linked to a 40-kDa polyethylene glycol (PEG) to prolong the circulation of the conjugates, was developed by Noxxon Pharma. NOXA12 is currently under development as a combination medication for several oncology therapies [44], especially focused on a phase I/II combination trial in metastatic pancreatic and colorectal cancer. It is believed that NOX-A12 fights against tumors in three ways: (1) breaking tumor protection to enable active immune cells, T cells, to infiltrate the tumor [45]; (2) blocking the bridge between the attraction of “repair cells” and the tumors; (3) mobilizing hidden tumor cells away from the bloodstream where tumor-killing drugs are more effective [46]. Since NOX-A12 is built from L-ribose units, it is therefore highly resistant to nuclease degradation. NOX-A12 binds and neutralizes CXCL12, a key chemokine (signaling) protein which promotes tumor proliferation and new blood vessel formation, and protects tumors from apoptosis [47]. It also inhibits CXCL12-induced chemotaxis of chronic lymphocytic leukemia (CLL) cells and sensitizes CLL cells toward bendamustine and fludarabine [48]. NOX-A12 also enhanced the infiltration of T cells which was synergistically activated by programmed death-1 (PD-1) blockade in the spheroids, indicating that the agents complement one another [49].

19.3.1.2 Aptamers Against Age-Related Macular Degeneration MACUGEN Macugen, also known as pegaptanib, consists of 28 nucleotides. It is the first commercialized RNA aptamer-based drug used in the treatment of wet age-related macular degeneration (AMD). Pegaptanib selectively binds to vascular endothelial growth factor (VEGF)-165, the VEGF isoform primarily responsible for pathological ocular neovascularization and vascular permeability [50]. The binding of 125I-labeled VEGF to human umbilical vein endothelial cells was inhibited by pegaptanib when cocultured, with an half maximal inhibitory concentration (IC50) of 0.75
1.4 nM [51]. The elimination half-lives of pegaptanib in rhesus monkeys were 9.3 and 12 h individually after a single intravenous or subcutaneous administration (1 mg/kg), respectively [52]. Population pharmacokinetic studies in patients with neovascular AMD [53] or diabetic macular edema [54] indicated pegaptanib did not accumulate in the plasma after multiple doses, and there was an insignificant impact of gender, race, hypertension, and glaucoma on the pharmacokinetic (PK) of pegaptanib.

19.3.1.3 Aptamers for Antithrombotic Therapy Thromboses are major determinants of morbidity and mortality in the elderly [55]. Heparin remains a crucial anticoagulant given its ability for binding and enhancing the inhibitory activity of the plasma protein antithrombin against blood-coagulation factors IIa (thrombin),

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IXa, and Xa [56]. However, heparin therapy was also associated with significant bleeding risks [57], and was consequently faced with the dilemma of the risk of thrombosis without treatment and the risk of bleeding due to treatment. One strategy is to pursue new medications to selectively target a specific coagulation protein. So far, several RNA aptamers [58] with different binding affinities (with Kd from 0.4 [59] to 13 nM [60]) to factor IXa (FXIa) and diverse mechanisms, including disrupting tissue factor VII(a) complex assembly [60], or interfering with factor XII and anionic binding [59] have been reported. Among these, reversible RNA aptamer antagonists (9.3t) [61] bound to factor XIa with a Kd of 0.58 nM exhibited greater than 5000-fold specificity for FXIa compared to the structurally similar coagulation factors VIIa, Xa, and XIa, and activated protein C. With respect to DNA aptamers, only two have been described to date. The ssDNA ligand 60-18 [29] inhibited thrombin-catalyzed fibrin clot formation with a Kd of approximately 0.5 nM [62]. The second, designated Factor ELeven Inhibitory APtamer strongly bound factor XIa with Kd of 1.8 nM and competitively inhibited factor XIa complex formation with antithrombin [63].

19.3.1.4 Aptamers Against Other Diseases Obesity is induced when excessive energy is deposited in the body and is related to the differentiation of white adipocytes [64]. Kim et al. isolated two aptamers (MA-33 and 91) that bound mature adipocyte cells 3T3-L1 with Kd of 143 and 33 nM individually but not preadipocytes [65]. Given the observation that inhibition of glucagon receptor (GCGR) activity is correlated with reduced glucose production in diabetes mellitus, Wang et al. reported a GCGR DNA aptamer antagonist GR-3 which specifically bound membrane protein of CHOGCGR cells with a Kd of 53 nM [66]. The presence of proteolytic autoantibodies against myelin basic protein often represents the occurrence of multiple sclerosis (MS). RNA Apt2-9 (of 71-nt length) with high affinity with Kd of 15 nM and specificity (28-fold Kd difference for specific and nonspecific binding) bound to proteolytic anti-myelin basic protein (MBP) autoantibodies from MS patients [67]. Covalent conjugate of the aptamer and Ca21-regulated photoprotein obelin further were developed into a bioluminescent microplate sensor to detect target antibodies [67].

19.3.2 Aptamer
Drug Conjugates for Targeted Therapies Classical methods for disease treatment, such as chemo-, radio-, photodynamic, and photothermal therapy can cause serious side effects in patients due to nonspecificity. Personalized, targeted therapy therefore has been gaining increasing attention. Targeted therapy aims to specifically maximize toxicity in diseased tissues while minimizing toxicity in healthy ones. Consequently, small cytotoxic molecules with specificity and selectivity must be considered during drug development, which can be achieved through medicinal chemistry optimization [68] or pharmaceutical formulation procedures [69]. In contrast, the development of drug
ligand conjugates could also fulfill targeted therapy. In these conjugates, the ligands specifically recognize disease-implicated receptors and deliver conjugated drugs to target cells but may not necessarily have direct therapeutic effects; drugs are conjugated to ligands via functional linkers that ensure the stability of conjugates and also allow conditional drug

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release in diseased cells. Among ligands for targeted therapy, antibodies are well established for specific recognition and/or biological regulation, hence antibody
drug conjugates have become an emerging novel class of antidisease treatment agents [70]. Alternatively, aptamers are of great interest for developing aptamer
drug conjugates (ApDCs) for targeted therapy. ApDCs, being a very simple but effective approach for disease treatment, often consist of a guidance (aptamer) and a warhead (drug) when noncovalently connected. Additional linker is needed if aptamer sequences are covalently joined with therapeutic agents. Beside aptamer
chemotherapeutic conjugates, aptamers also enable targeted delivery of nanoparticles and drug-containing carriers through the conjugation process, or other drugs, toxins, or photosensitizers to targeted cells, in particular cancer tissues [71], by implementing prodrug strategies.

19.3.2.1 Aptamer
Chemotherapeutic Conjugates Chemotherapy is one of the most common treatments for diseases, in particular doxorubicin (Dox) for cancer therapy. Dox is a commercial oncology drug commonly used to treat breast cancer, bladder cancer, Kaposi’s sarcoma, lymphoma, and acute lymphocytic leukemia. However, its therapy was often compromised by side effects because of undistinguished cell toxicity of many chemotherapeutics. The side effects of Dox included loss/thinning of hair, degeneration of bone marrow, nausea and vomiting, inflammation in the mouth and lips, which were often observed mainly due to their limited selectivity and the consequent “offtarget” effects. Thanks to the ability of current technologies to design and site-specifically modify aptamers with wanted functional groups, aptamers are subject to conjugate with Dox, both noncovalently and covalently. DNA aptamer sgc8 selectively targets protein tyrosine kinase 7, which is overexpressed in many types of cancer [72]. Hence, Dox
sgc8c conjugates with different strategies and their therapies have been investigated incrementally. For instance, aptamer sgc8c was coupled with Dox through a pH liable hydrazone linker which can be cleaved inside the acidic cancer cell environment (Fig. 19
4A). This conjugates not only showed the high binding affinity (Kd 5 2.0 6 0.2 nM) and the capability to be efficiently internalized into target cells which inherited from sgc8c aptamer (Fig. 19
4B), but also excellent specificity for binding target CCRF
CEM cancer cells [73]. Despite the proof of concept of targeted chemotherapeutics delivery through ApDCs, the main challenge to this strategy is low copy number of drug conjugated onto each aptamer (1:1 ratio), which may not thoroughly exert the potential of the aptamer. To increase the ratio of Dox
aptamer, Zhu et al. reported synthetic drug
DNA adducts (Fig. 19
4C) in which multiple copies of Dox were site-specifically conjugated on deoxyguanosine of each sgc8c through the methylene that linked the 3-NH2 group of Dox on one side to the 2-NH2 of deoxyguanosine on the other side [74]. Beside maintaining the potency of Dox toward cancer cells, this conjugate also offer targeted Dox delivery, with exceptional resistance to nuclease degradation, temperature-induced Dox release, programmability, and robustness of design [74]. Except pH- and temperature-liable linkage of aptamer and drug, photocleavable linker nitrobenzene was also introduced to bridge sgc8 with multiple copies of the anticancer drug fluorouracil [75].

FIGURE 19–4 Aptamer
chemotherapeutic covalent conjugates. (A) Conjugation of the drug Dox to aptamer sgc8c. (B) Binding assay of sgc8c
Dox conjugates to CCRF
CEM cells. (C) Schematic illustration of nuclease-resistant synthetic drug
DNA adducts. (D) Physical-conjugate formation between an aptamer and a drug molecule, Dox. (E) Schematic diagram of ApDC intercalation. ApDC, Aptamer
drug conjugate; Dox, doxorubicin. (A) Reproduced with permission from Y.F. Huang, D. Shangguan, H. Liu, J.A. Phillips, X. Zhang, Y. Chen, et al., Molecular assembly of an aptamer-drug conjugate for targeted drug delivery to tumor cells, ChemBioChem 10 (5) (2009) 862
868. © 2009, Wiley-VCH. (B) Reproduced with permission from Y.F. Huang, D. Shangguan, H. Liu, J.A. Phillips, X. Zhang, Y. Chen, et al., Molecular assembly of an aptamer-drug conjugate for targeted drug delivery to tumor cells, ChemBioChem 10 (5) (2009) 862
868. © 2009, WileyVCH. (C) Reproduced with permission from G. Zhu, S. Cansiz, M. You, L. Qiu, D. Han, L. Zhang, et al., Nuclease-resistant synthetic drug-DNA adducts: programmable drug-DNA conjugation for targeted anticancer drug delivery, NPG Asia Mater. 7 (3) (2015) e169. © 2015, Springer Nature. (D) Reproduced with permission from V. Bagalkot, O.C. Farokhzad, R. Langer, S. Jon, An aptamer
doxorubicin physical conjugate as a novel targeted drug-delivery platform, Angew. Chem. Int. Ed. 45 (48) (2006) 8149
8152. © 2006, Wiley-VCH. (E) Reproduced with permission from G. Yu, H. Li, S. Yang, J. Wen, J. Niu, Y. Zu, ssDNA aptamer specifically targets and selectively delivers cytotoxic drug doxorubicin to HepG2 cells, PLoS One 11 (1) (2016) e0147674. © 2016, PLoS One, open access.

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The ApDCs through chemical modifications of the drugs and aptamer may to a greater or lesser extent affect the safety and efficacy profile of the drugs and the binding characteristics of the aptamer, thereby resulting in less efficacious ApDCs. It would be desirable to develop simple but effective targeted drug-delivery strategies that do not require chemical modification of the drug or/and aptamer. Aptamers are known to form tertiary conformations with short double-stranded regions through intramolecular base pairing, while the planar aromatic chromophore of the molecule intercalates between two base pairs of the DNA [76] and inhibits macromolecular biosynthesis in Dox-exposed cells. It was therefore assumed that Dox may intercalate into the double-stranded regions and form a physical complex with the aptamers, contributing to a noncovalent stacking interaction without modification of the drug or aptamer. The purpose of this mechanism is to offer Dox a unique advantage to noncovalently formulate ApDCs since the aptamer stem part enables the preferred insertion of fused aromatic fragments into tandem 50 -(GC)-30 or 50 -(CG)-30 sites. Noncovalent ApDCs are instinctively believed to maintain the inherent profiles of drug or the aptamer to the greatest extent possible. In 2006 Bagalkot et al. first reported this strategy (Fig. 19
4D) for the targeted delivery of Dox to cancer cells through the formation of prostate-specific membrane antigen aptamer A10 RNA-Dox physical conjugate [77]. The continuation of this pioneering work engineered aptamers with additional drug-intercalating sites, attempting to increase the drug-loading capacity while maintaining the recognition abilities of aptamers. To intercalate more Dox molecules, HepG2-specific aptamer was modified with paired CG repeats at the 50 -end, leading to four Dox molecules (mol/mol) that were fully intercalated (Fig. 19
4E) in each conjugate aptamer-Dox (ApDC) molecule [78]. Alternatively, a long GC tail was tethered to the 50 end of aptamer TLS11a to generate a modified aptamer TLS11a-GC. TLS11a-GC forms a dimer structure which could theoretically increase the TLS11a-to-Dox ratio from 1:2 to 1:28, simultaneously fulfilling the targeted delivery to liver cancer cells [79].

19.3.2.2 Targeted Drug-Delivery Vehicles Conjugated With Aptamers Beside covalent or noncovalent conjugates of aptamers with chemotherapeutics for drug delivery and release, more robust and multifunctional aptamers involved in detection/ delivery vehicles toward disease cells have also been fabricated. By combining originally lowaffinity aptamers with micelles, Wu et al. obtained a self-assembled aptamer
micelle nanostructure (Fig. 19
5A) with selective and strong binding at physiological conditions [80]. Unlike the above-mentioned aptamer
chemotherapeutic conjugates, the aptamer
micelle is supposed to deliver drug to target cells without internalization of the aptamer’s target molecule. However, they can simply interact with the cell membrane and quickly release the doped hydrophobic drug molecules into the cells. In addition, by replacing PEG of this aptamer
micelle vehicle with therapeutic aptamer plus linker, the heterogeneous aptamer
micelle can specifically deliver, for example, aptamer drugs such as Macugen around the target cell surface [80]. Liposomes were widely used as drug-delivery materials because of their reduced toxicity and the enhanced stability of drugs by encapsulation.

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FIGURE 19–5 Drug-delivery vehicles conjugated with aptamers. (A) Aptamer
micelle. (B) Aptamer
liposomes. (A) Reprinted with permission from Y. Wu, K. Sefah, H. Liu, R. Wang, W. Tan, DNA aptamer
micelle as an efficient detection/delivery vehicle toward cancer cells, Proc. Natl. Acad. Sci. U.S.A. 107 (1) (2010) 5
10. © 2010 National Academy of Sciences. (B) Adapted from L. Li, J. Hou, X. Liu, Y. Guo, Y. Wu, L. Zhang, et al., Nucleolin-targeting liposomes guided by aptamer AS1411 for the delivery of siRNA for the treatment of malignant melanomas, Biomaterials 35 (12) (2014) 3840
3850.

Functionalization of Dox-encapsulated liposomes with nucleotide aptamers gave rise to aptamosomes which turned out to be superior to the intercalation of Dox into the prostatespecific membrane antigen-specific RNA aptamer in specificity and Dox delivery efficiency toward prostate-specific membrane antigen (PSMA)(1) cancer cells [81]. AS1411, a commercialized aptamer-specific binding to nucleolin, was conjugated to PEGylated cationic liposome (Fig. 19
5B) as the targeting probe AS1411-PEG-liposome (ASLP) which reacted with anti-BRAF siRNA (siBraf) through electrostatic interaction. The ASLP/siRNA complex strongly silenced the activity of the malignant melanoma BRAF gene and had much higher accumulation of the siRNA in tumor cells compared with normal cells [82].

19.3.3 Biosensors Made From Aptamers Biosensors are analytical vessels in which sensing reactions depend upon biomolecular recognition, and are used to detect the appearance or concentration of a biological analyte. Biosensors consist of three elements: a biological recognition element that recognizes the analyte and produces a signal, a signal transducer, and a reader. Unlike other spectraoriented analytical methods, which typically undergo sequential pretreatments in one analysis, biosensors are easy-to-operate analytical devices [83]. Aptamer-based sensors combining an aptamer as the biosensing element integrated on the transducer surface has been coined “aptasensors.” Aptasensors takes advantage of the high affinity and tunability of aptamers, while their sensitivity is remarkably affected by the transducer. Here, we review some typical aptasensor applications.

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19.3.3.1 Electrochemical Detection Electrochemical aptasensors have been paid greater attention due to their high sensitivity, fast response, robustness, minituarization potential, and various targets (e.g., small molecules, macromolecules, and cells) [84]. Their applications for food and environmental contaminant detection [85], safeguarding [86], and quality [87] have been recently reviewed. Examples of electrochemical aptasensors include the detection of tetracycline (TC) in milk by combination of biosensor 1 fabricated from ferrocene and biosensor 2 prepared from carbon nanofibers and AuNP nanocomposite. The as-prepared aptasensors detect tetracycline (TET) quantitatively in the range of 1028
1023 g/L, with a detection limit of 3.3 3 1027 g/L, which is in complete agreement with the results determined by ultrahigh-performance liquid chromatography
tandem mass spectrometry [88]. It turned out that aptasensors are effective devices for sensitive and selective monitoring of toxins, including ochratoxin A (OTA) [89]. An aptasensor was designed as an OTA aptamer, with two AuNPs modified with probe 1 and probe 2 to form a Y-shaped DNA duplex with disassembly AuNP dimers by the OTA (Fig. 19
6A). This reverse process of the assembly of AuNP dimers in the presence of OTA led to the color of the aqueous solution of AuNPs turning from blue to red and enabled the accurate colorimetric detection of OTA concentration as 5.17 nM, a result highly consistent with the result obtained from a commercial enzyme-linked immunosorbent assay (ELISA) kit [90]. In addition to the detection of small ligands, probably the real advantage of aptasensors is in recognizing macromolecules. By combining bare gold film and apatamer
graphene oxide (AGO) [91], Lou constructed a sandwich surface plasmon resonance (SPR) detection immunoassay of prion disease-associated isoform (PrPSc), which gave both qualitative and quantitative information (Fig. 19
6B). The detection sensitivity of AGO for PrPSc was B156fold higher than SPR direct detection format [91]. Cytokines are signaling proteins, such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α, secreted by immune cells in order to regulate the immune response and may be used to diagnose infectious diseases. Liu et al. developed micropatterned aptamer-modified electrodes for simultaneous detection of TNF-α and IFN-γ [92]. To enable multiplexing, IFN-γ and TNF-α aptamers were labeled with anthraquinone and methylene blue redox reporters, respectively, and fixed on gold, therefore producing a redox signal which depends upon the concentration of target cytokines. The electrodes were further integrated into microfluidic devices and used to dynamically monitor cytokine release from immune cells [93]. To enhance the sensitivity of IFN-γ detection, a nanostructured electrode was created using silicon nanowires (NWs) which were covered with gold and further functionalized with thiolated aptamers specific for IFN-γ; the NW aptasensors responded three times faster and were twice as sensitive to IFN-γ compared to standard flat electrodes. Most significantly, NW aptasensors allowed detection of IFN-γ from as few as 150 T cells/mL (Fig. 19
6C), while ELISA did not pick up a signal from the same number of cells [94].

19.3.3.2 Optical Detection The presence of a specific ligand can be detected by aptamers since hybridization/interaction between an aptamer and its target may produce a signal which can be optically

FIGURE 19–6 Aptasensors for electrochemical detection. (A) Small molecules of ochratoxin A. (B) Protein. (C) T cell. (A) Adapted from R. Xiao, D. Wang, Z. Lin, B. Qiu, M. Liu, L. Guo, et al., Disassembly of gold nanoparticle dimers for colorimetric detection of ochratoxin A, Anal. Methods 7 (3) (2015) 842
845. (B) Adapted from Z. Lou, J. Wan, X. Zhang, H. Zhang, X. Zhou, S. Cheng, et al., Quick and sensitive SPR detection of prion disease-associated isoform (PrP Sc) based on its self-assembling behavior on bare gold film and specific interactions with aptamer-graphene oxide (AGO), Colloids Surf, B: Biointerfaces 157 (2017) 31
39. (C) Adapted from Y. Liu, A. Rahimian, S. Krylyuk, T. Vu, B. Crulhas, G. Stybayeva, et al., Nanowire aptasensors for electrochemical detection of cell-secreted cytokines, ACS Sens. 2 (11) (2017) 1644
1652.

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detected, such as by fluorescence, chemiluminescence, colorimetry, surface-enhanced Raman scattering spectroscopy, and SPR. For an overview of other optical aptasensor detection methods, refer to Ref. [95]. This chapter focuses on the discussion of SPR since it is one of the methods to select and characterize aptamers [96], however it is relatively less exploited and importantly a label-free optical detection platform. SPR biosensors can be applied in aptasensing platforms by immobilizing an aptamer on a selective surface. Notwithstanding, the greatest challenge to SPR-based aptasensors is their sensitivity, in particular for small molecules [97]. Therefore sensitivity improvement has become the focus of this type of device. It has been shown that the addition of gold nanoparticles, such as nanorods [98], will increase the sensitivity of an SPR-based aptasensor. In a recent study, Bianco et al. reported that the sensitivity of the aptasensor depended on the aptamer immobilization strategy: the limit of detection (LOD) of self-assembled monolayers or direct immobilization (0.005 ng/mL) (Fig. 19
7A) is threefold higher than that of mixed or in the presence of an aliphatic spacer immobilization (0.020 ng/mL) approach [99]. It was reported that gold nanostars (GNSs) can enhance aptamer
antibody(anti-TC) detection for the analysis of TC based on an SPR assay: an LOD of 10 Attomolar could be detected using

FIGURE 19–7 SPR-based aptasensors. (A) Aptasensor direct connection and through aliphatic spacer to the surface and mixed. (B) Conjugation of a gold nanostar with an aptasensor. SPR, Surface plasmon resonance. (A) Adapted from M. Bianco, A. Sonato, A. De Girolamo, M. Pascale, F. Romanato, R. Rinaldi, et al., An aptamer-based SPRpolarization platform for high sensitive OTA detection, Sens. Actuators, B: Chem. 241 (2017) 314
320. (B) Reproduced with permission from S. Kim, H.J. Lee, Gold nanostar enhanced surface plasmon resonance detection of an antibiotic at attomolar concentrations via an aptamer-antibody sandwich assay, Anal. Chem. 89 (12) (2017) 6624
6630. Copyright from ACS 2017.

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GNS-anti-TC (Fig. 19
7B), which was .103-fold higher than when using anti-TC only [100]. Thanks to the contribution of microfluidic chip removal impurity, a gold nanorodfunctionalized SPR-based aptasensor was able to sense the release of cytochrome-c with an LOD of 0.1 ng/mL due to the anticancer drug effects [101].

19.3.3.3 Other Detection Methods It is believed that ligand/aptamer binding induced surface stress changes. The microcantilever works on mechanical displacements exerted by the interaction of ligand and analyte. Based on this understanding, Kang et al. immobilized a cocaine-specific aptamer in a microcantilever using the interferometric technique (Fig. 19
8A). Due to binding induced by surface stress of cocaine with aptamer, the sensitivity of the cocaine concentration was determined by adding from 25 to 500 μM and surface stress values of cocaine with aptamer from 11 to 26 mN/m were obtained, which are proportional to the concentration of cocaine [102].

FIGURE 19–8 Aptasensors to detect: (A) surface stress and (B) electronic signal. (A) Reproduced with permission from K. Kang, A. Sachan, M. Nilsen-Hamilton, P. Shrotriya, Aptamer functionalized microcantilever sensors for cocaine detection, Langmuir 27 (23) (2011) 14696
14702. Copyright from ACS 2011. (B) Reproduced with permission from Y. Ohno, K. Maehashi, K. Matsumoto, Label-free biosensors based on aptamer-modified graphene field-effect transistors, J. Am. Chem. Soc. 132 (51) (2010) 18012
18013. Copyright from ACS 2010.

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Field-effect transistors are transistors where the electric field influences the output current. A biological sensing single layer of graphene is required in this application for better sensing. Aptamer-modified graphene-FET (Fig. 19
8B) was used to monitor the target IgE protein at different concentrations, which was reflected by electrical signal drain current fluctuations [103]. In another study, single-walled carbon nanotube field effect transistor (SWCNT-FET), where the thrombin aptamer is immobilized in the side wall of SWCNT-FET, the addition of thrombin caused an abrupt decrease in conductance and the LOD observed was 10 nM [104]. The use of aptamers in the lateral flow technology as an alternative to antibodies has great advantages [105]. It is very cost effective and can be produced in a large scale. Qin et al. established a class of strip biosensors based on thrombin aptamer-linked gold nanoparticle aggregates, which occur cracking reaction when the target recognized its homologous aptamer. Combined theaptamer-cleavage reaction with the enzyme catalytic amplification system, the authors claimed that their lateral flow strip biosensor enable visual detection of 6.4 pM thrombin without instrumentation within 12 minutes [106].

19.3.4 Diagnostic Applications Medical diagnoses based on molecular features can be highly specific and extremely sensitive when the correct recognition molecule and an efficient signal transduction system are employed. These aptamer-based systems are nonimmunogenic and nontoxic [107], the immobilization of aptamers is very easy compared to antibodies as aptamers can be synthesized chemically, and they can be modified easily at their 30 or 50 end with biotin, primary amino, or other functional groups, which makes them more advantageous [108]. Diagnostics based on aptamers is a promising emerging platform, in this study the author detected zeptomolar concentrations of protein, which is a 1000-fold increase in sensitivity compared with conventional ELISA [109]. The first use of a diagnostic tool with aptamers was in 1999 for Bacillus anthracic spores to detect anthrax spore [110]. It is used in a microscopic technique where a fluorescein isothiocyanate-conjugated aptamer is used as a novel tumor marker in tissue sections of rat brain glioblastoma (GBM) to selectively visualize the paths and the branching of the neoangiogenetic, pathologic microvasculature [111]. In cytological application, to detect GBM, EGFR, the most common oncogene in GBM with anti-EGFR RNA aptamers, could capture both human and murine GBM cells [112].

19.4 Conclusion and Future Perspective The aptamer, also called chemical antibody, is polynucleotides with low molecular weight and often identified through high-throughput screening methods such as SELEX. It can specifically recognize biological targets with high affinity. Similar to antibody aptamers, their primary biological function is targeting. However, unlike antibody, aptamers can be chemically synthesized once their sequence is determined, paving the way for large-scale manufacturing. Polymeric aptamers exist with specific structures and recognize their target

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through noncovalent interactions. Since aptamers are made of only four types of nucleotides, they are easy to design and assemble following predictable thermodynamics, which is an incomparable advantage over antibodies in the authors’ opinion. In addition, they can be easily modified [113] with little or no compromise in their biological roles, which offer aptamers a great opportunity to conjugate or combine with other components, such as chemotherapeutics for targeted therapies, vehicles for drug delivery, nanomaterials for biosensing [114], and detection of interested analytes based on different methods and disease diagnostics. Compared with polypeptidic antibody, aptamer is still in an immature stage of development given the width and depth of study needed, let alone commercialized products. These underdevelopments may be attributed to the following. (1) Understanding the bias of nucleotides themselves since it is widely accepted that they function as genetic material, while other functions of polynucleotides are only just being appreciated. (2) Although aptamers alone can work as therapeutics, fully exploration of their functions depend upon combinations with other materials/components, therefore modification of aptamers to a certain degree is inevitable. However, the cost of the chemical modification enabling conjugation can be 10 times higher than native ones [115] according to our experience, which is a major challenge for the broader investigation of aptamers. (3) The unsolved stability of aptamers sets a heavy barrier to their successful application due to the abundance of nucleases, which again implies the necessity of hybrid aptamers with suitable stabilizers. Although unexploited, aptamer-related investigations have already verified their role in theranostic biomaterials. We are also confident that aptamer-oriented researches are going to increase rapidly and that aptamer-based products will enter clinics in the near future.

Acknowledgments The authors acknowledge financial support from the Academy of Finland (Decision No. 297580), Jane and Aatos Erkko Foundation (Grant No. 4704010), and Sigrid Jusélius Foundation (Decision No. 28001830K1).

References [1] A.D. Ellington, J.W. Szostak, In vitro selection of RNA molecules that bind specific ligands, Nature 346 (6287) (1990) 818. [2] C. Tuerk, L. Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science 249 (4968) (1990) 505
510. [3] Y. Li, A quarter century of in vitro selection, J. Mol. Evol. 81 (5-6) (2015) 137
139. [4] H. Jo, C. Ban, Aptamer
nanoparticle complexes as powerful diagnostic and therapeutic tools, Exp. Mol. Med. 48 (5) (2016) e230. [5] W.E. Wright, M. Binder, W. Funk, Cyclic amplification and selection of targets (CASTing) for the myogenin consensus binding site, Mol. Cell. Biol. 11 (8) (1991) 4104
4110. [6] C. Chai, Z. Xie, E. Grotewold, SELEX (Systematic Evolution of Ligands by EXponential Enrichment), as a powerful tool for deciphering the protein
DNA interaction space, Plant Transcr. Factors, 754, 2011, pp. 249
258.

Chapter 19 • Nucleotide Aptamers as Theranostic Biomaterials

441

[7] H. Hasegawa, N. Savory, K. Abe, K. Ikebukuro, Methods for improving aptamer binding affinity, Molecules 21 (4) (2016) 421. [8] K.-T. Guo, G. Ziemer, A. Paul, H.P. Wendel, CELL-SELEX: novel perspectives of aptamer-based therapeutics, Int. J. Mol. Sci. 9 (4) (2008) 668
678. [9] S. Ma, N. Tang, J. Tian, DNA synthesis, assembly and applications in synthetic biology, Curr. Opin. Chem. Biol. 16 (3) (2012) 260
267. [10] R.A. Beckman, L.M. Weiner, H.M. Davis, Antibody constructs in cancer therapy, Cancer 109 (2) (2007) 170
179. [11] D.V. Tassev, N.-K.V. Cheung, Monoclonal antibody therapies for solid tumors, Expert Opin. Biol. Ther. 9 (3) (2009) 341
353. [12] R. Dienstmann, B. Markman, J. Tabernero, Application of monoclonal antibodies as cancer therapy in solid tumors, Curr. Clin. Pharmacol. 7 (2) (2012) 137
145. [13] P.J. Bates, D.A. Laber, D.M. Miller, S.D. Thomas, J.O. Trent, Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer, Exp. Mol. Pathol. 86 (3) (2009) 151
164. [14] X. Ni, M. Castanares, A. Mukherjee, S.E. Lupold, Nucleic acid aptamers: clinical applications and promising new horizons, Curr. Med. Chem. 18 (27) (2011) 4206
4214. [15] R.E. Wang, H. Wu, Y. Niu, J. Cai, Improving the stability of aptamers by chemical modification, Curr. Med. Chem. 18 (27) (2011) 4126
4138. [16] V. Thiviyanathan, D.G. Gorenstein, Aptamers and the next generation of diagnostic reagents, Proteomics Clin. Appl. 6 (11-12) (2012) 563
573. [17] Y. Zhang, J. Tu, D. Wang, H. Zhu, S.K. Maity, X. Qu, et al., Programmable and multifunctional DNAbased materials for biomedical applications, Adv. Mater. 30 (2018) e1703658. [18] J. Zhou, J. Rossi, Aptamers as targeted therapeutics: current potential and challenges, Nat. Rev. Drug Discov. 16 (2017) 181
202. [19] J.A. Capra, K. Paeschke, M. Singh, V.A. Zakian, G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae, PLoS Comput. Biol. 6 (7) (2010) e1000861. [20] J. Da Costa, Catalytic Potential and Ligand Binding Properties of the Malachite Green RNA Aptamer, University of Waterloo, 2008. [21] Q. Zhang, R. Landgraf, Selecting molecular recognition. What can existing aptamers tell us about their inherent recognition capabilities and modes of interaction? Pharmaceuticals 5 (5) (2012) 493
513. [22] M. Cho, S.S. Oh, J. Nie, R. Stewart, M. Eisenstein, J. Chambers, et al., Quantitative selection and parallel characterization of aptamers, Proc. Natl. Acad. Sci. U.S.A. 110 (46) (2013) 18460
18465. [23] T. Gerling, K.F. Wagenbauer, A.M. Neuner, H. Dietz, Dynamic DNA devices and assemblies formed by shape-complementary, non-base pairing 3D components, Science 347 (6229) (2015) 1446
1452. [24] W. Bi, X. Bai, F. Gao, C. Lu, Y. Wang, G. Zhai, et al., DNA-templated aptamer probe for identification of target proteins, Anal. Chem. 89 (7) (2017) 4071
4076. [25] Y. Wang, R.R. Rando, Specific binding of aminoglycoside antibiotics to RNA, Chem. Biol. 2 (5) (1995) 281
290. [26] M.G. Wallis, U. von Ahsen, R. Schroeder, M. Famulok, A novel RNA motif for neomycin recognition, Chem. Biol. 2 (8) (1995) 543
552. [27] V. Tereshko, E. Skripkin, D.J. Patel, Encapsulating streptomycin within a small 40-mer RNA, Chem. Biol. 10 (2) (2003) 175
187. [28] S.B. Long, M.B. Long, R.R. White, B.A. Sullenger, Crystal structure of an RNA aptamer bound to thrombin, RNA 14 (12) (2008) 2504
2512.

442

THERANOSTIC BIONANOMATERIALS

[29] A. Pica, I. Russo Krauss, V. Parente, H. Tateishi-Karimata, S. Nagatoishi, K. Tsumoto, et al., Throughbond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers, Nucleic Acids Res. 45 (1) (2017) 461
469. [30] M. Davlieva, J. Donarski, J. Wang, Y. Shamoo, E.P. Nikonowicz, Structure analysis of free and bound states of an RNA aptamer against ribosomal protein S8 from Bacillus anthracis, Nucleic Acids Res. 42 (16) (2014) 10795
10808. [31] C. Gao, J. Desaphy, M. Vieth, Are induced fit protein conformational changes caused by ligand-binding predictable? A molecular dynamics investigation, J. Comput. Chem. 38 (15) (2017) 1229
1237. [32] S.-J. Choi, C. Ban, Crystal structure of a DNA aptamer bound to PvLDH elucidates novel single-stranded DNA structural elements for folding and recognition, Sci. Rep. 6 (2016) 34998. [33] W.A. Kibbe, OligoCalc: an online oligonucleotide properties calculator, Nucleic Acids Res 35 (Suppl. 2) (2007) W43
W46. [34] J. Zuber, H. Sun, X. Zhang, I. McFadyen, D.H. Mathews, A sensitivity analysis of RNA folding nearest neighbor parameters identifies a subset of free energy parameters with the greatest impact on RNA secondary structure prediction, Nucleic Acids Res. 45 (10) (2017) 6168
6176. [35] M. van Dijk, A.M. Bonvin, 3D-DART: a DNA structure modelling server, Nucleic Acids Res. 37 (Suppl. 2) (2009) W235
W239. [36] J. Caroli, C. Taccioli, A. De La Fuente, P. Serafini, S. Bicciato, APTANI: a computational tool to select aptamers through sequence-structure motif analysis of HT-SELEX data, Bioinformatics 32 (2) (2015) 161
164. [37] G. Van Zundert, J. Rodrigues, M. Trellet, C. Schmitz, P. Kastritis, E. Karaca, et al., The HADDOCK2.2 web server: user-friendly integrative modeling of biomolecular complexes, J. Mol. Biol. 428 (4) (2016) 720
725. [38] I. Jeddi, L. Saiz, Three-dimensional modeling of single stranded DNA hairpins for aptamer-based biosensors, Sci. Rep. 7 (1) (2017) 1178. [39] A.T. Catherine, S.N. Shishido, G.A. Robbins-Welty, A. Diegelman-Parente, Rational design of a structureswitching DNA aptamer for potassium ions, FEBS Open Bio 4 (1) (2014) 788
795. [40] J. Zhu, H. Huang, S. Dong, L. Ge, Y. Zhang, Progress in aptamer-mediated drug delivery vehicles for cancer targeting and its implications in addressing chemotherapeutic challenges, Theranostics 4 (9) (2014) 931
944. [41] Y. Cho, Y.B. Lee, J.-H. Lee, D.H. Lee, E.J. Cho, S.J. Yu, et al., Modified AS1411 aptamer suppresses hepatocellular carcinoma by up-regulating galectin-14, PLoS One 11 (8) (2016) e0160822. [42] E.M. Reyes-Reyes, Y. Teng, P.J. Bates, A new paradigm for aptamer therapeutic AS1411 action: uptake by macropinocytosis and its stimulation by a nucleolin-dependent mechanism, Cancer Res. 70 (21) (2010) 8617
8629. [43] Y. Cheng, G. Zhao, S. Zhang, F. Nigim, G. Zhou, Z. Yu, et al., AS1411-induced growth inhibition of glioma cells by up-regulation of p53 and down-regulation of Bcl-2 and Akt1 via nucleolin, PLoS One 11 (12) (2016) e0167094. [44] A. Vater, S. Klussmann, Turning mirror-image oligonucleotides into drugs: the evolution of Spiegelmers therapeutics, Drug Discov. Today 20 (1) (2015) 147
155. [45] D.T. Fearon, The carcinoma-associated fibroblast expressing fibroblast activation protein and escape from immune surveillance, Cancer Immunol. Res. 2 (3) (2014) 187
193. [46] R. Marasca, R. Maffei, NOX-A12: mobilizing CLL away from home, Blood 123 (7) (2014) 952
953. [47] K.S. Jeng, C.J. Jeng, W.J. Jeng, C.F. Chang, I. Sheen, Role of CXC chemokine ligand 12/CXC chemokine receptor 4 in the progression of hepatocellular carcinoma, Oncol. Lett. 14 (2) (2017) 1905
1910.

Chapter 19 • Nucleotide Aptamers as Theranostic Biomaterials

443

[48] J. Hoellenriegel, D. Zboralski, C. Maasch, N.Y. Rosin, W.G. Wierda, M.J. Keating, et al., The Spiegelmer NOX-A12, a novel CXCL12 inhibitor, interferes with chronic lymphocytic leukemia cell motility and causes chemosensitization, Blood 123 (7) (2014) 1032
1039. [49] D. Zboralski, K. Hoehlig, D. Eulberg, A. Frömming, A. Vater, Increasing tumor-infiltrating T cells through inhibition of CXCL12 with NOX-A12 synergizes with PD-1 blockade, Cancer Immunol. Res. 5 (11) (2017) 950
956. [50] E.W. Ng, D.T. Shima, P. Calias, E.T. Cunningham, D.R. Guyer, A.P. Adamis, Pegaptanib, a targeted antiVEGF aptamer for ocular vascular disease, Nat. Rev. Drug Discov. 5 (2) (2006) 123
132. [51] C. Bell, E. Lynam, D.J. Landfair, N. Janjic, M.E. Wiles, Oligonucleotide NX1838 inhibits VEGF165mediated cellular responses in vitro, In Vitro Cell. Dev. Biol. Anim. 35 (9) (1999) 533
542. [52] C.E. Tucker, L.-S. Chen, M.B. Judkins, J.A. Farmer, S.C. Gill, D.W. Drolet, Detection and plasma pharmacokinetics of an anti-vascular endothelial growth factor oligonucleotide-aptamer (NX1838) in rhesus monkeys, J. Chromatogr. B Biomed. Sci. Appl. 732 (1) (1999) 203
212. [53] A.S. Basile, M. Hutmacher, D. Nickens, J. Nielsen, K. Kowalski, L. Whitfield, et al., Population pharmacokinetics of pegaptanib in patients with neovascular, age-related macular degeneration, J. Clin. Pharmacol. 52 (8) (2012) 1186
1199. [54] A.S. Basile, M.M. Hutmacher, K.G. Kowalski, K.Y. Gandelman, D.J. Nickens, Population pharmacokinetics of pegaptanib sodium (Macugens) in patients with diabetic macular edema, Clin. Ophthalmol. 9 (2015) 323. [55] W.H. Geerts, D. Bergqvist, G.F. Pineo, J.A. Heit, C.M. Samama, M.R. Lassen, et al., Prevention of venous thromboembolism: American College of Chest Physicians evidence-based clinical practice guidelines, Chest 133 (Suppl. 6) (2008) 381S
453S. [56] E. Gray, J. Hogwood and B. Mulloy, The anticoagulant and antithrombotic mechanisms of heparin. In: Lever R., Mulloy B., Page C. (eds) Heparin - A Century of Progress, Handb. Exp. Pharmacol., 207, 2012, pp. 43
61. [57] R. Fujiwara, A. Yoshida, A. Takei, K. Fukuzawa, K. Takami, M. Takami, et al., “Heparin bridging” increases the risk of bleeding complications in patients undergoing anticoagulation therapy and device implantation, J. Arrhythm. 28 (2) (2012) 96
99. [58] S.K. Buddai, J.M. Layzer, G. Lu, C.P. Rusconi, B.A. Sullenger, D.M. Monroe, et al., An anticoagulant RNA aptamer that inhibits proteinase-cofactor interactions within prothrombinase, J. Biol. Chem. 285 (8) (2010) 5212
5223. [59] R. Woodruff, Y. Xu, J. Layzer, W. Wu, M. Ogletree, B. Sullenger, Inhibiting the intrinsic pathway of coagulation with a factor XII-targeting RNA aptamer, J. Thromb. Haemost. 11 (7) (2013) 1364
1373. [60] C.P. Rusconi, A. Yeh, H.K. Lyerly, J.H. Lawson, B.A. Sullenger, Blocking the initiation of coagulation by RNA aptamers to factor VIIa, Thromb. Haemost. 84 (5) (2000) 841
848. [61] C.P. Rusconi, E. Scardino, J. Layzer, G.A. Pitoc, T.L. Ortel, D. Monroe, et al., RNA aptamers as reversible antagonists of coagulation factor IXa, Nature 419 (6902) (2002) 90
94. [62] D.M. Tasset, M.F. Kubik, W. Steiner, Oligonucleotide inhibitors of human thrombin that bind distinct epitopes, J. Mol. Biol. 272 (5) (1997) 688
698. [63] D.A. Donkor, V. Bhakta, L.J. Eltringham-Smith, A.R. Stafford, J.I. Weitz, W.P. Sheffield, Selection and characterization of a DNA aptamer inhibiting coagulation factor XIa, Sci. Rep. 7 (2017) 2102. [64] D. Hausman, M. DiGirolamo, T. Bartness, G. Hausman, R. Martin, The biology of white adipocyte proliferation, Obes. Rev. 2 (4) (2001) 239
254. [65] E.Y. Kim, J.W. Kim, W.K. Kim, B.S. Han, S.G. Park, B.H. Chung, et al., Selection of aptamers for mature white adipocytes by cell SELEX using flow cytometry, PLoS One 9 (5) (2014) e97747. [66] G. Wang, J. Liu, K. Chen, Y. Xu, B. Liu, J. Liao, et al., Selection and characterization of DNA aptamer against glucagon receptor by cell-SELEX, Sci. Rep. 7 (1) (2017) 7179.

444

THERANOSTIC BIONANOMATERIALS

[67] M.A. Vorobjeva, V.V. Krasitskaya, A.A. Fokina, V.V. Timoshenko, G.A. Nevinsky, A.G. Venyaminova, et al., RNA aptamer against autoantibodies associated with multiple sclerosis and bioluminescent detection probe on its basis, Anal. Chem. 86 (5) (2014) 2590
2594. [68] L. Yan, C. Yan, K. Qian, H. Su, S.A. Kofsky-Wofford, W.-C. Lee, et al., Diamidine compounds for selective inhibition of protein arginine methyltransferase 1, J. Med. Chem. 57 (6) (2014) 2611
2622. [69] M.M. Lü btow, L. Hahn, M.S. Haider, R. Luxenhofer, Drug specificity, synergy and antagonism in ultrahigh capacity poly(2-oxazoline)/poly(2-oxazine) based formulations, J. Am. Chem. Soc. 139 (32) (2017) 10980
10983. [70] N. Diamantis, U. Banerji, Antibody-drug conjugates—an emerging class of cancer treatment, Br. J. Cancer 114 (4) (2016) 362
367. [71] H. Sun, X. Zhu, P.Y. Lu, R.R. Rosato, W. Tan, Y. Zu, Oligonucleotide aptamers: new tools for targeted cancer therapy, Mol. Ther. Nucleic Acids 3 (2014) e182. [72] O. Jacobson, I.D. Weiss, L. Wang, Z. Wang, X. Yang, A. Dewhurst, et al., 18F-Labeled single-stranded DNA aptamer for PET imaging of protein tyrosine kinase-7 expression, J. Nucl. Med. 56 (11) (2015) 1780
1785. [73] Y.F. Huang, D. Shangguan, H. Liu, J.A. Phillips, X. Zhang, Y. Chen, et al., Molecular assembly of an aptamer-drug conjugate for targeted drug delivery to tumor cells, ChemBioChem 10 (5) (2009) 862
868. [74] G. Zhu, S. Cansiz, M. You, L. Qiu, D. Han, L. Zhang, et al., Nuclease-resistant synthetic drug-DNA adducts: programmable drug-DNA conjugation for targeted anticancer drug delivery, NPG Asia Mater. 7 (3) (2015) e169. [75] R. Wang, G. Zhu, L. Mei, Y. Xie, H. Ma, M. Ye, et al., Automated modular synthesis of aptamer
drug conjugates for targeted drug delivery, J. Am. Chem. Soc. 136 (7) (2014) 2731
2734. [76] D. Agudelo, P. Bourassa, G. Bérubé, H.-A. Tajmir-Riahi, Intercalation of antitumor drug doxorubicin and its analogue by DNA duplex: structural features and biological implications, Int. J. Biol. Macromol. 66 (2014) 144
150. [77] V. Bagalkot, O.C. Farokhzad, R. Langer, S. Jon, An aptamer
doxorubicin physical conjugate as a novel targeted drug-delivery platform, Angew. Chem. Int. Ed. 45 (48) (2006) 8149
8152. [78] G. Yu, H. Li, S. Yang, J. Wen, J. Niu, Y. Zu, ssDNA aptamer specifically targets and selectively delivers cytotoxic drug doxorubicin to HepG2 cells, PLoS One 11 (1) (2016) e0147674. [79] L. Meng, L. Yang, X. Zhao, L. Zhang, H. Zhu, C. Liu, et al., Targeted delivery of chemotherapy agents using a liver cancer-specific aptamer, PLoS One 7 (4) (2012) e33434. [80] Y. Wu, K. Sefah, H. Liu, R. Wang, W. Tan, DNA aptamer
micelle as an efficient detection/delivery vehicle toward cancer cells, Proc. Natl. Acad. Sci. U.S.A. 107 (1) (2010) 5
10. [81] H. Park, D.M. Kim, S.E. Baek, K.S. Kim, D.E. Kim, Comparison of drug delivery efficiency between doxorubicin intercalated in RNA aptamer and one encapsulated in RNA aptamer-conjugated liposome, Bull. Korean Chem. Soc. 36 (10) (2015) 2494
2500. [82] L. Li, J. Hou, X. Liu, Y. Guo, Y. Wu, L. Zhang, et al., Nucleolin-targeting liposomes guided by aptamer AS1411 for the delivery of siRNA for the treatment of malignant melanomas, Biomaterials 35 (12) (2014) 3840
3850. [83] S. Chandra, J. Chapman, A. Power, J. Roberts, D. Cozzolino, The application of state-of-the-art analytic tools (biosensors and spectroscopy) in beverage and food fermentation process monitoring, Fermentation 3 (4) (2017) 50. [84] K. Han, T. Liu, Y. Wang, P. Miao, Electrochemical aptasensors for detection of small molecules, macromolecules, and cells, Rev. Anal. Chem. 35 (4) (2016) 201
211. [85] R. Rapini, G. Marrazza, Electrochemical aptasensors for contaminants detection in food and environment: recent advances, Bioelectrochemistry 118 (2017) 47
61.

Chapter 19 • Nucleotide Aptamers as Theranostic Biomaterials

445

[86] G.K. Mishra, V. Sharma, R.K. Mishra, Electrochemical aptasensors for food and environmental safeguarding: a review, Biosensors 8 (2) (2018) 28. [87] A. Vasilescu, J.-L. Marty, Electrochemical aptasensors for the assessment of food quality and safety, TrAC Trends Anal. Chem. 79 (2016) 60
70. [88] Q. Xu, Z. Liu, J. Fu, W. Zhao, Y. Guo, X. Sun, et al., Ratiometric electrochemical aptasensor based on ferrocene and carbon nanofibers for highly specific detection of tetracycline residues, Sci. Rep. 7 (1) (2017) 14729. [89] H.B. Bostan, N.M. Danesh, G. Karimi, M. Ramezani, S.A.M. Shaegh, K. Youssefi, et al., Ultrasensitive detection of ochratoxin A using aptasensors, Biosens. Bioelectron. 98 (2017) 168
179. [90] R. Xiao, D. Wang, Z. Lin, B. Qiu, M. Liu, L. Guo, et al., Disassembly of gold nanoparticle dimers for colorimetric detection of ochratoxin A, Anal. Methods 7 (3) (2015) 842
845. [91] Z. Lou, J. Wan, X. Zhang, H. Zhang, X. Zhou, S. Cheng, et al., Quick and sensitive SPR detection of prion disease-associated isoform (PrP Sc) based on its self-assembling behavior on bare gold film and specific interactions with aptamer-graphene oxide (AGO), Colloids Surf, B: Biointerfaces 157 (2017) 31
39. [92] Y. Liu, T. Kwa, A. Revzin, Simultaneous detection of cell-secreted TNF-α and IFN-γ using micropatterned aptamer-modified electrodes, Biomaterials 33 (30) (2012) 7347
7355. [93] Y. Liu, Z. Matharu, A. Rahimian, A. Revzin, Detecting multiple cell-secreted cytokines from the same aptamer-functionalized electrode, Biosens. Bioelectron. 64 (2015) 43
50. [94] Y. Liu, A. Rahimian, S. Krylyuk, T. Vu, B. Crulhas, G. Stybayeva, et al., Nanowire aptasensors for electrochemical detection of cell-secreted cytokines, ACS Sens. 2 (11) (2017) 1644
1652. [95] S.H. Jalalian, N. Karimabadi, M. Ramezani, K. Abnous, S.M. Taghdisi, Electrochemical and optical aptamer-based sensors for detection of tetracyclines, Trends Food Sci. Technol. 73 (2018) 45
57. [96] E. Dausse, A. Barré, A. Aimé, A. Groppi, A. Rico, C. Ainali, et al., Aptamer selection by direct microfluidic recovery and surface plasmon resonance evaluation, Biosens. Bioelectron. 80 (2016) 418
425. [97] Z. Zhu, M. Feng, L. Zuo, Z. Zhu, F. Wang, L. Chen, et al., An aptamer based surface plasmon resonance biosensor for the detection of ochratoxin A in wine and peanut oil, Biosens. Bioelectron. 65 (2015) 320
326. [98] S. Balamurugan, K.M. Mayer, S. Lee, S.A. Soper, J.H. Hafner, D.A. Spivak, Nanostructure shape effects on response of plasmonic aptamer sensors, J. Mol. Recognit. 26 (9) (2013) 402
407. [99] M. Bianco, A. Sonato, A. De Girolamo, M. Pascale, F. Romanato, R. Rinaldi, et al., An aptamer-based SPR-polarization platform for high sensitive OTA detection, Sens. Actuators, B: Chem. 241 (2017) 314
320. [100] S. Kim, H.J. Lee, Gold nanostar enhanced surface plasmon resonance detection of an antibiotic at attomolar concentrations via an aptamer-antibody sandwich assay, Anal. Chem. 89 (12) (2017) 6624
6630. [101] J.F.-C. Loo, P.-M. Lau, S.-K. Kong, H.-P. Ho, An assay using localized surface plasmon resonance and gold nanorods functionalized with aptamers to sense the cytochrome-c released from apoptotic cancer cells for anti-cancer drug effect determination, Micromachines 8 (11) (2017) 338. [102] K. Kang, A. Sachan, M. Nilsen-Hamilton, P. Shrotriya, Aptamer functionalized microcantilever sensors for cocaine detection, Langmuir 27 (23) (2011) 14696
14702. [103] Y. Ohno, K. Maehashi, K. Matsumoto, Label-free biosensors based on aptamer-modified graphene field-effect transistors, J. Am. Chem. Soc. 132 (51) (2010) 18012
18013. [104] H.-M. So, K. Won, Y.H. Kim, B.-K. Kim, B.H. Ryu, P.S. Na, et al., Single-walled carbon nanotube biosensors using aptamers as molecular recognition elements, J. Am. Chem. Soc. 127 (34) (2005) 11906
11907.

446

THERANOSTIC BIONANOMATERIALS

[105] A. Chen, S. Yang, Replacing antibodies with aptamers in lateral flow immunoassay, Biosens. Bioelectron. 71 (2015) 230
242. [106] C. Qin, W. Wen, X. Zhang, H. Gu, S. Wang, Visual detection of thrombin using a strip biosensor through aptamer-cleavage reaction with enzyme catalytic amplification, Analyst 140 (22) (2015) 7710
7717. [107] E.S. Group, Anti-vascular endothelial growth factor therapy for subfoveal choroidal neovascularization secondary to age-related macular degeneration: phase II study results, Ophthalmology 110 (5) (2003) 979
986. [108] S. Balamurugan, A. Obubuafo, S.A. Soper, D.A. Spivak, Surface immobilization methods for aptamer diagnostic applications, Anal. Bioanal. Chem. 390 (4) (2008) 1009
1021. [109] E. Baldrich, A. Restrepo, C.K. O'Sullivan, Aptasensor development: elucidation of critical parameters for optimal aptamer performance, Anal. Chem. 76 (23) (2004) 7053
7063. [110] J.G. Bruno, J.L. Kiel, In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection, Biosens. Bioelectron. 14 (5) (1999) 457
464. [111] M. Blank, T. Weinschenk, M. Priemer, H. Schluesener, Systematic evolution of a DNA aptamer binding to rat brain tumor microvessels selective targeting of endothelial regulatory protein pigpen, J. Biol. Chem. 276 (19) (2001) 16464
16468. [112] Y. Wan, Y.T. Kim, N. Li, S.K. Cho, R. Bachoo, A.D. Ellington, et al., Surface-immobilized aptamers for cancer cell isolation and microscopic cytology, Cancer Res. 70 (22) (2010) 9371
9380. [113] T. Ö sterlund, H. Korhonen, P. Virta, DNA-templated N (Me)-alkoxyamine glycosylation, Org. Lett. 20 (6) (2018) 1496
1499. [114] L. Yang, X. Zhang, M. Ye, J. Jiang, R. Yang, T. Fu, et al., Aptamer-conjugated nanomaterials and their applications, Adv. Drug Delv. Rev. 63 (14-15) (2011) 1361
1370. [115] Z. Zhuo, Y. Yu, M. Wang, J. Li, Z. Zhang, J. Liu, et al., Recent advances in SELEX technology and aptamer applications in biomedicine, Int. J. Mol. Sci. 18 (10) (2017) 2142.

20 Electronic Structures of Alkaline Rare Earth Fluoride-Based Upconversion Nanomaterials Bolong Huang DEPART ME NT OF APPLIED B IOLOGY AND CHEMICAL TECHNOLOGY, THE HONG KONG POL YTE CHNIC UNI VERSIT Y, K OWL OON, HONG K ONG S AR, P.R. C HINA

20.1 Introduction Powerful and longer upconversion (UC) luminescence with flexible wavelength is increasingly needed for biochemical and materials engineering applications. Lanthanide (Ln) materials have been recognized to be the optimal host system for UC luminescence due to their fluoride system having a lower rate of photo-induced charge carrier recombination and higher efficiency for energy transfer (ET) than other rare earth (RE) materials. Presently, terminating the quenching sites for nanosized UC luminescence materials is a significant ET efficiency challenge. Chemical doping is effective to modify the electronic structure and surface property of UC luminescence nanoparticles (UCNPs), but will potentially import new extrinsic impurity levels to block the inter-Ln31 transfer channels. Recently, a phosphor luminescence technique assisted by core
shell nanostructured Ln fluoride with RE has been leading on modulating the UC luminescence properties. However, accurately controlled defect engineering in the core
shell UCNPs is difficult under current nanotechnologies because most modulation techniques still rest on the conventional experimental routine through a combination of host materials and activating dopant ions without solid theoretical support and guidance. Experimentally, UC luminescence can be modulated through effective enhancement for original emissions and multicolor output regulation. The designated activators and core
shell structural engineering will decide the optimal combinations and Ln activators determine the output wavelength. The intensity and efficiency of UC luminescence are governed by the suppression of surface quenching states. Unfortunately, the relationship between excitation energy migration dynamics and their UC luminescence property modulation is still unclear, especially at the excited electronic levels. Very few works on the ab initio calculation, large-scale, and substantial efforts concentrate on both experimental synthesis and efficient calculation for future exploration. Therefore it is necessary to apply density Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00020-1 © 2019 Elsevier Inc. All rights reserved.

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functional theory (DFT) for systematic investigation of the quenching sites and interface band offset engineering for future material modulation engineering. From a theoretical angle, Ln spectroscopy is strongly influenced by the relative locations of both Ln and intrinsic defect levels in the band gap and the 4f fine-levels re-alignment must rest on the lowest binding energy levels of the Ln dopant. One of our collaborators (Prof. Pieter Dorenbos) developed and demonstrated that the overall excitation levels for both divalent and trivalent Ln ions within core
shell can be confirmed if the lowest binding energies are confirmed, which is based on systematic studies on Ln doping levels for over 10,000 solid materials. However, the current theoretical DFT method suffers from limited accuracy because the main electron binding energies in Ln and host band levels obtained from provided empirical models do not give accurate self-consistent determination on the Hubbard-type Coulomb repulsion energies (Hubbard-U) within the 4f
4f exchangecorrelated interaction. The determined optical band gap widths are far from experimental data results. Due to the nephelauxetic effects, electrons excited to a higher level at the Ln dopant center will substantially interact with intrinsic defect levels from the host lattice of UCNPs. Also, low accuracy on native point defect levels and doping impurity levels will give the wrong physicochemical trend in level-matching induced resonant ET between intrinsic levels and 4f fine-levels. Moreover, the intrinsic defect levels of the host lattice will usually overlap with the Ln doping levels, acting as an energetic depleting channel. The closed-shell singularity issue in conventional single-way linear response determination in current DFT can be overcome by our two-way crossover linear response method to search the Hubbard-U. This first-principle DFT 1 U calculation without empirical parameters will further benefit us on the electronic structures. The electronic structural engineering of the UCNPs will be possible with systematic investigation on the inherent principles. The computational issues are another impeding challenge for providing reasonable theoretical support for high-efficiency UC luminescence material design. With these corner-stone works, we can use the band offset to tune the relative location of excitation levels in the band gap to achieve UC luminescence engineering. Furthermore, the interface band offset can ameliorate the potential overlap issue between deep intrinsic defect levels and UC excitation levels and, thus, the energy depletion channels can be suppressed. The RE-based fluoride system has become the optimal host system for UC luminescence due to a lower rate of photo-induced charge carrier recombination and higher UC ET efficiency [1,2]. However, for the UCNPs, the surface effect turns out to be stronger than the bulk effect for the Ln UCNPs due to the large surface-to-volume ratio, which causes a greater quenching effect for Ln UCNPs [3]. The overall downgrading of the luminescence intensity is mainly attributed to the surface quenching effect that affects both the ET between luminescence centers in the near-surface region and the activation centers in the nanoparticles (NPs). Therefore extensive studies to discover the quenching prohibition and related mechanism is the key to increasing the UC luminescence efficiency. The core
shell engineered NPs with RE ion-assisted phosphor luminescence technique was originally imported for enhancing the UC efficiency with a higher quantum yield [1] and

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has aroused tremendous interests in biological, chemical, and physical applications [4
13]. As has been confirmed [2,14
18], a uniformly epitaxial grown shell over the core is necessary to effectively block the ET from activator (RE-dopant ion) sites in the core to surface defects, capping ligands, and solvent molecules [15,18
23]. With developed synthesis, the shell can also accommodate RE-ion doping and efficiently transfer the energy from the core to the shell to modulate the color [20,21] or intensity of output UC luminescence [14,22]. Accordingly, core
shell nanostructural engineering introduces a new variable (core
shell recombination) into UC materials design. In general, a designated synthesis with selective chemical doping is an effective strategy to modify the electronic structure and surface properties of UCNPs to exhibit significantly improved performance [4
6,15,24]. In detail, the specific synthesis conditions, the appropriate tuned dopant/trap levels within single- or multishell [20
22] via layer-by-layer epitaxial growth, precisely controlled shell thickness or surface coating in the core
shell nanostructure [25
29] have been attempted to control the required output UC luminescent properties. The perfect epitaxial growth of layer-by-layer multishell [29] can be distinguished from the core via various matured experimental techniques [16,17,23,26]. For the impact from shell thickness [25,26,30], both homogeneous and heterogeneous shell can passivate the surface quenching state of the core effectively [18]. Nevertheless, with recent gradual concentration on the interface [31
34], an exact theoretical guiding mechanism of ET at the interface for UC luminescence in the core
shell structure is still not perfectly explained and established. Some primarily important works by van Veggel et al. [16,17] support that a decent systematic investigation on the topics of interface ET and ionic exchange/diffusion etc. [31
34] is required since the quantum yields of core
shell systems are still constrained and the role of doped ions is under dispute. In addition, the heterojunction induced band offsets always occur at the interface in terms of either multilayered core
shell systems or thin film stacks. The band offsets of the two host systems are controlled by such interface dipoles [35
38]. The interface usually has dipoles formed by charge transfer across the bond at the interface. Such contributions on ET in both radiative [39,40] and nonradiative ET [41,42] should be also accounted for together in Auzel’s ET UC (ETU) model [40] in a core
shell system. As described by Auzel [39] and Anderson [43], respectively, for an ETU model of UC, it is still not fully clarified whether the charge carrier transport across the interface is exactly responsible for energy transferred via nonradiative transmission. Moreover, due to the “Ln contraction” [44], the screening constant of the shielding effect for 4f electrons of Ln is large but less than one, partially treating the 4f in electronic interactions with valence shells is necessary. According to the partially screened 4f electrons on the valence shell, the contributions of 4f states in the band structures of host solids are significant and cannot be neglected. As energy barriers for spatially separated charges, the interface-induced band offsets also influence the transfer. This will happen when the on-site-trapped electrons are also responsible for upconverted energy transfer to 4f electrons at higher excited states with the process of multiphoton absorption. Therefore it is necessary to predict the absolute locations of the defect levels and 4f energy levels of Ln relative to the valence and conduction bands (CBs) and this has been also discussed in our preliminary DFT calculations [45].

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There are many routes for ETs of UC luminescence with single or combined processes (excited state absorption [ESA], ETU, etc.) in Ln31-doped UCNPs. As pointed out by Yan et al., there is no universal mechanism for any of the luminescent Ln31 ions [18]. Some recent novel discoveries have raised interesting questions about previously established models. For instance, an experiment shows that Ce31 cannot effectively sensitize Sm31, Dy31, and Eu31 but Tb31 was attributed to the redox process between these S
A pairs [46], which means there was a substantial valence exchange among them and leads to a deduction that 4f valence electrons are no longer exactly shielded. This is obviously distinct from the review by Liu et al. [14] or other groups [18]. Therefore experiments still cannot explain some UC processes with an ET model on the basis of a single-ion level. Moreover, the question will be also applicable for the following discussion on both cores and shells, particularly the interface. Furthermore, Liu et al. [22] proposed that the accurate description of energy levels within the host band structures in both the ETU and the energy migration-mediated UC (EMU) process is also highly necessary. The prerequisite is the correct determination of the lowest levels, the ground-state electronic levels within the band gap for such optical materials. In systematic works done by our collaborators [47,48], such ground-state levels of Ln31 ions are located within the band gap of host solids and their excited states are even overlaps with the band structures from valence to CBs. Therefore the host matrix should be considered as the first priority in estimating the quantum efficiency of the UC luminescence, especially the interface of such core
shell NPs. Since the core
shell interface is still to be explored and discussed, a comprehensive interface DFT calculation is needed as a solid reference for future engineering of the UC luminescence in RE core
shell nanostructures. Some theoretical methods have successfully been applied to predict the luminescence properties, including luminescence decay and excitation migration of Ln ions in crystal structures [49,50]. However, current firstprinciples calculations based on empirical parameters [51
53] result in nearly 11% [54] error electronic structures with the experiments for NaYF4 and NaGdF4. In addition, the 4f electronic fine levels have evidently been underestimated, with a relative error of 30%. Theoretical study remains at the semiempirical stage and accurate prediction of the 4f electronic excited- and ground-state levels related to the optical band gap still faces challenges and opportunities [55]. To improve the accuracy on excitation energies, theoretic DFT works are still insufficient, with much potential to be developed. Zunger and Freeman [56] applied a self-consistent numerical basis set within local-density formalism to describe the excitation states of LiF as total-energy differences between (separately calculated) excitedand ground-state configurations. Later, Janak [57] introduced an occupation number that is equal to the eigenvalue of the orbital to generalize the total energy for certain excitation state calculations. The most recent work of Ku et al. [58] developed LDA 1 U within timedependent DFT which provided a detailed discussion of Frenkel excitons with reasonable accuracy. Hence, developing a DFT calculation method with higher accuracy in luminescence materials will be highly desired in combination with experimental techniques on luminescence engineering.

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On the other hand, recent developments UCNPs still leave us the challenge of continuously decent understanding about the energy migration crossover in the interface between the core and shell region of NaLuF4 (Ln 5 Y, Gd and Lu) core-shell nanocrystals (NCs). As we know, the core
shell interfaces are mostly constructed by F
Ln (Ln 5 Y, Gd, Lu) or F
Na bonds. The interface states induced by local structures of F sites inevitably affect or quench the UC luminescence of electronic excitations among 4f orbitals by RE31 ions. The role of alkalineions, such as Na or Li, may have less influence on the electronic structure as their s-orbital electrons are usually far below the Fermi level. But for ionic conductions, they actually are good charge carriers with relatively low barriers for long-range ordered lattice transport. Thus the UCNPs are the most complicated regions to understand, and are not only for ET between interions, but also an electronic transition for energy conversion by excitations. As stated above, though β-NaYF4 is a prominent host material for fluorescence phosphor and UC luminescence, theoretical electronic structure study has not been well reported and remains at an initial stage such as levels of local density formalism. This arises because its atomic structure presents a degree of disorder for atomic site occupancy of different elements like Na and Y. In addition, the local atomic structures of F sites are significant to the structural thermal stability (which determines phonon vibration modes) and electronic structures for highly efficient optical transitions of fluorescence. As is known, the 2p orbitals of all F sites line up the top part of the valence band (VB) of NaLnF4 (Ln 5 Y, Gd, and Lu). Meanwhile, the VB maximum (VBM) behavior is dominated by the 2p orbitals of F sites and is modulated by various locally disordered F sites. Compared to oxides, the sensitive electronic structures may be due to the more evident charge transfer between different cations and their connected F sites.

20.2 Calculation Setup As to our ab initio determination of the Hubbard U on orbitals, the geometries and lattice parameters of all Ln sesquioxides were optimized using Perdew-Burke-Ernzerhof (PBE) functional calculations. This procedure reduces the computational cost and ensures the reliability of the Hubbard U value obtained by our self-consistent iterative calculations. We use this procedure before the Hubbard U determination because DFT has been already verified as reliable for the structural optimization of compound solids with 4f or 5f orbitals [59], even with ultrasoft pseudopotentials. This may be due to the well-developed pseudopotential technique and, more importantly, to the fact that f-electrons have a small influence on the lattice parameters when treated as valence electrons, as shown by the small difference in the DFT and DFT 1 U calculated lattice parameters [45,60
63]. Moreover, the U parameter will be determined more carefully through our developed self-consistent method [45,60
63]. Realistic fluoride coordination in P-6 UCNPs like β-NaLnF4 (Ln 5 Y, Gd, and Lu) could be very different. The CASTEP code is used to perform our DFT 1 U calculations [64]. The Na, Y/Gd, and F norm-conserving pseudopotentials are generated using the OPIUM code in the Kleinman
Bylander projector form [65], and the nonlinear partial core correction [66] and a

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THERANOSTIC BIONANOMATERIALS

scalar relativistic averaging scheme [67] are used to treat the spin-orbital coupling effect. In particular, we treated the 4f, 5s, 5p, 5d, 6s states as the valence states of the Gd atoms. The Rappe-Rabe-Kaxiras-Joannopoulos (RRKJ) method is chosen for the optimization of the pseudopotentials [68]. The general gradient correction type functional (e.g., PBE) was chosen to combine the rotational invariant approximated Hubbard-U correction term [69] on designated f and d orbitals with a kinetic cutoff energy of 750 eV. Their valence shells are expanded in a plane-wave basis set. The ensemble DFT method of Marzari et al. [70] is used for convergence. Reciprocal space integration was performed using k-point grids of 8 3 8 3 4k points in the β-NaYF4 Brillouin zone (BZ). For all of the electronic state calculations in NaGdF4, we use the self-consistent determination for the U correction on the localized 4f orbitals to correct the on-site Coulomb energy of the electron spurious self-energy. In previous work, we have established a manner to determine the on-site electronic self-energy and related wavefunction relaxation in the orbitals, so as to obtain accurate orbital eivenvalues for electronic structures [45,71,72]. The detail process was referred to in the previous work. With our self-consistent determination process, the on-site Hubbard U parameters for 4f of Gd and different 2p of F sites are obtained shown in Table 20
1, respectively. For the pseudopotentials of the Gd used for calculation, we similarly chose a nonlinear core correction technique for correcting the valencecore charge density overlapping in such heavy fermion elements, the detail discussion of this method is presented in a previous work about the native point defect study of CeO2 [62,63]. The valence electron configurations for the pseudopotentials we chose for the generation were 5d14f7 for Gd21 as the ground-state configuration. All of the DFT 1 U calculations were performed on the norm-conserving pseudopotential theoretical scheme. This will help us to reflect all-electron behavior of the valence electrons, especially for the subtle effect of the 4f electrons and outer 6s electrons. Table 20–1 Summary of Lattice Constants, Electronic Band Gap, Hubbard Orbital Potential Corrections for Different Sites for β-NaYF4, β-NaGdF4, β-NaLuF4

a (Å) c (Å) Eg (eV) Ud-Y1 (eV) Ud-Y2 (eV) Uf-Gd1 (eV) Uf-Gd2 (eV) Uf-Lu1 (eV) Uf-Lu2 (eV) Up-F1 (eV) Up-F2 (eV) Up-F3 (eV)

β-NaYF4 Model 1

Model 2

β-NaGdF4 Model 1

Model 2

β-NaLuF4 Model 1

Model 2

6.146 7.127 8.492 4.241 5.278



4.447 4.227 4.264

6.120 7.168 8.500 4.257 4.782



4.215 4.421 4.161

6.175 7.274 7.690

9.065 2.708

4.360 4.243 4.196

6.154 7.334 7.406

2.314 11.916

4.278 4.483 4.500

6.003 6.936 8.811



12.273 7.349 4.091 4.059 4.069

5.981 6.962 8.849



7.989 15.346 4.094 4.186 4.056

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20.3 Results and Discussions We introduce the electronic structures of β-NaLnF4 (Ln 5 Y, Gd, and Lu) NCs and represent them by two different local lattice arrangements following P-6 symmetry. This can generate the effect of various coordinated F sites. Based on this method, we classify the mentioned two types of lattices in forms of variations of local coordination number (CN) for F sites. The first model (model 1) has F coordination of 3, 4, and 5 differently, but the weighted average number is kept at 4. Meanwhile the second one (model 2) has a CN of F exactly at 4, but has bonding with different neighboring ions with bonding angles distorted. By using the ground-state formation enthalpy (ΔHf) calculations: ΔHf 5 μ(NaLnF4)μ(Na)-μ(Ln)-4μ(F), model 2 is found to be relatively more stable than model 1, which means such a nanocrystal structure energetically prefers the local structure with CN of F exactly at 4. (Note: The above μ is their calculated total energies.) The energy difference has a magnitude of 37.9 meV. The above energy difference confirms the physical trend that even two types of lattice share the same symmetry, while they still can be distinguished through local structural motifs as some extent of local F redistribution could allow more energy to be relaxed. Detailed differences in the structures have been demonstrated in Fig. 20
1. We can see in

FIGURE 20–1 (A) Lattice model 1 for β-NaYF4 (Na 5 purple, Y 5 cyan, and threefold-F 5 pink, fourfold-F 5 red, and fivefold-F 5 blue). (B) Local structure of fivefold F sites with nearest neighboring Na and Y sites. The related first BZ is shown. (C) Lattice model 2 for β-NaYF4 (Na 5 purple, Y 5 cyan, and fourfold-F 5 red). (D) Local structure of fourfold F sites with nearest neighboring Na and Y sites. The related first BZ is shown. BZ, Brillouin zone.

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THERANOSTIC BIONANOMATERIALS

model 1, the indicated F sites that connect with two Y/Gd/Lu and three Na atoms have CN 5 5, acting like a distorted octahedral F site. Model 2 gives only one tetrahedral type coordinated F site (i.e., fourfold coordination) existing in the lattice, with two connecting with Y sites and two with Na sites, respectively. Thus the contrast originates from the intermediate local F CN between Na-(Y/Gd/Lu) layers.

20.4 β-NaYF4 From the band structures (Fig. 20
2A and B), two different NaYF4 models in P-6 symmetry have nearly the same band gaps, which are 8.492 and 8.500 eV for the one containing distorted octahedral F sites and the one with tetrahedral F sites, respectively. Their VB widths are nearly the same, at 3.08 and 3.31 eV, respectively, which is largely contributed by the 2p orbitals of F sites. The energy range of the lower parts of the CBs has been leveled up by the 4d orbitals of Y sites with the nearly the same widths as 3.51 eV. The CB minimums of both structures are located at the Γ point of the first BZ. However, the structure with distorted octahedral F sites has an indirect band gap. The direct transition gap of Γ!Γ is 8.532 eV, which is very close to the experimental measured value of 8.5 eV. The indirect transitions gaps of K!Γ and M!Γ are 8.503 and 8.492 eV, respectively.

20.5 β-NaGdF4 We now look at the β-NaGdF4 system, which is a good charge transfer insulator for UC luminescence. Similarly, we use model 1 and 2 for comparison. Experimentally, the uniform growth of core
shell for NaGdF4 is difficult to reach [17]. In analogy with this observation, we simulate it to consist of two different local lattice presentations within P-6 symmetry as models 1 and 2. The following discussion shows that different local symmetries generate the 4f orbital potentials of three Gd sites in β-NaGdF4 which are not equivalent to each other

FIGURE 20–2 Band structure and total DOS of β-NaYF4 in model 1 (A) and model 2 (B). DOS, Density of state.

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455

and are split into two different types. First, in model 1, Gd1 and Gd2 sites are ordinary halffilled shells since they have 4f7 configuration in which the self-energy is counteracted by orbital relaxation, while Gd3 reflects an open shell effect like 4f72δ (δ . 0). Model 2 also shows similar effects, as shown in Fig. 20
3. This indicates that two different β-NaGdF4 models always have two different types of Gd31 ions, which are half-filled and open shells for 4f orbitals, respectively. Therefore the contributions given by Gd ions on the optical transitions and luminescence regarding the energy transport should be considered separately, as these are two different types of Gd ions in the β-NaGdF4 lattice system. The Gd sites are found to be easily distributed on the surface region of the core
shell nanocrystals [17]. We can see that model 1 is slightly denser than model 2 for β-phase close to the experiments. Therefore the core part is likely to be model 1 where the Gd sites are flexibly coordinated by F sites. Since the surface region is actually an area that the stress

FIGURE 20–3 Hubbard orbital potential corrections on 4f of Gd1 and Gd2 sites in model 1 (A), Gd3 in model 1 (B), Gd1 in model 2 (C), and Gd2 and Gd3 in model 2 (D). (UR and USE are the Hubbard projections for orbital relaxation and self-energy functionals. The Δ is the residue when UR is subtracted from USE. U4f is the normalized Hubbard U parameter for DFT 1 U calculations with minimized residue.) DFT, Density functional theory.

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THERANOSTIC BIONANOMATERIALS

stably releases, it is appropriate for the model 2 lattice with an amount of tetrahedral coordinated F sites. Thus the shell can be simulated in the model 2 lattice. The energy-transfer efficiency is high through the Gd in the core to the activator ion in the shell part via another Gd also in the shell region [17,22]. According to Fig. 20
3, such highly efficient cross-section EMU energy migration is accomplished by two different Gd ions with two different energy levels and the low-lying one at the shell is acting as a relay center to take up the energy from the Gd ion in the core. However, from Fig. 20
4, the β-NaYF4 host lattice makes no contribution to this. The electronic structures of β-NaGdF4 turn to spin-polarization with some extent of ferromagnetic behavior (Fig. 20
5A and B). The spin moment is 7.19 per cell and is contributed by the Gd sites. The band gap is very similar to β-NaYF4. The 4f-empty states of Gd sites are found in the band gap. This reduces the energies of optical vertical interlevel transition from 2p!5d to 2p!4f. The two different 4f-filled states remain at 5.4 and 2.2 eV below the VBM for model 1, respectively.

FIGURE 20–4 Hubbard orbital potential correction on 4d of Y sites in model 1 (A), model 2 (B), as well as the 2p of F sites in model 1 (C), and model 2 (D). (UR and USE are the Hubbard projections for orbital relaxation and selfenergy functionals. Δ is the residue when UR is subtracted from USE. The crossover in (C) and (D) shows that the self-energy is counteracted by the orbital relaxation under the perturbation in the linear response method. U4d and U2p are the normalized Hubbard U parameter for DFT 1 U calculations with minimized residue.) DFT, Density functional theory.

Chapter 20 • Electronic Structures of Alkaline Rare Earth

457

FIGURE 20–5 Band structure and total DOS of β-NaGdF4 in model 1 (A) and model 2 (B). DOS, Density of state.

Model 2 has three different 4f-filled states, such as 1.7, 2.2, and 6.9 eV below the VBM. These 4f-filled states all present spin-up alignment in the projected partial density of states (PDOSs), while the 4f-empty states are all spin-down. The 4f-empty states of model 1 sit at 7.8 and 10.6 eV above VBM and 7.5 and 12.0 eV for model 2, respectively. The energy interval between the filled and empty states of 4f orbitals is 10.02 eV for model 1 and 9.54 eV for model 2. From the calculated band structures, the 4f-occupied level has split into two different levels and the first is 2.2 eV below the VBM with spin-up in these two models. The lowest 4f-unoccupied level is about 7.5 eV above the VBM with spin-down configuration. The PDOS calculation also shows the difference of evident splitting of 4f levels of Gd sites (Fig. 20
6). It implies the nonequivalent roles of three Gd sites in a P-6 symmetry cell. The band gap of model 1 is thus found to decrease to 7.690 eV by such an effect, and model 2 has an even lower gap of 7.406 eV.

20.6 β-NaLuF4 The case of β-NaLuF4 has a slightly difference to that of NaYF4 but similar to NaGdF4. Fig. 20
7A and B show that the Lu sites in the β-NaLuF4 with varied coordinated F sites all present an inert close shell effect, although they have different variation behavior with relation to the external Coulomb potential energies. However, Fig. 20
7C and D show that the Lu sites in β-NaLuF4 with all tetrahedral coordinated F sites are separated into two different classes as found in β-NaGdF4. The Lu1 site has an open shell of 4f orbitals which means the 4f14 of Lu1 is no longer inert. The 4f electrons will participate with other valence electrons like 5d16s2 together to bind with F sites. Lu2 and Lu3 show a close shell indicating inert 4f electron behavior in the β-NaLuF4 lattice. Therefore combining Figs. 20
3 and 20
7, it indicates that the semicore 4f orbitals of Lu are no longer inert acting as 4f142δ (δ . 0). This is true because the screening constants of 4f

458

THERANOSTIC BIONANOMATERIALS

FIGURE 20–6 Partial DOS of 2p, 4f, and 5d orbitals of β-NaGdF4 in model 1 and model 2 lattices. DOS, Density of state.

orbitals of Lns are relatively large but still less than one, which is why the “Ln contraction” occurs [44]. Our previous theoretical work also confirms this [45]. Electronic structure calculations (Fig. 20
8) on these two P-6 lattices of β-NaLuF4 are illustrated. The VB structures demonstrate an energetic difference at K (
1/3, 2/3, 0) and M (0 1/2 0) within reciprocal space. The VBM is found to be intermediate between K and M instead, which consist of F-2p orbitals in the local structure with a high F CN. This feature vanishes in the tetrahedral-like F coordinated lattice. Further, on the total density of states, the structure with high F CN has a relatively higher 4f-filled state at EV 5.8 eV, where EV denotes the position of VBM. The local structure with tetrahedral-like F coordination gives deeper filled states at EV 7.2 eV. Further, to look at the contributions of individual orbital components (PDOS; Fig. 20
9), the difference arises from the different 5d orbital positions of Lu sites. As we know, the CB in β-NaLuF4 is mostly contributed by 5d orbitals of the Lu ion. The calculated band width of 5d orbitals is around 2.5 eV, in agreement with trend. Different electronic structures show varied charge transfer behaviors. As model 1, it gives three distinct peaks

Chapter 20 • Electronic Structures of Alkaline Rare Earth

459

FIGURE 20–7 Hubbard orbital potential correction on 4f of Lu1 and Lu2 sites in model 1 (A), Lu3 in model 1 (B), Lu1 in model 2 (C), and Lu2 and Lu3 in model 2 (D). (UR and USE are the Hubbard projections for orbital relaxation and self-energy functionals. Δ is the residue when UR is subtracted from USE. U4f is the normalized Hubbard U parameter for DFT 1 U calculations with minimized residue.) DFT, Density functional theory.

FIGURE 20–8 Band structure and total DOS of β-NaLuF4 in model 1 (A) and model 2 (B). DOS, Density of state.

denoting that three different types of coordinated F sites induce three different 2p
5d charge transfer paths. In model 2, there are three different short-range orders varying with equal probability. Thus it has a nearly zero gradient of most CB levels given by 5d orbitals, which means the 5d levels of Lu sites are less distinctive in this lattice. Therefore

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THERANOSTIC BIONANOMATERIALS

FIGURE 20–9 Partial DOS of 2p, 4f, and 5d orbitals of β-NaLuF4 in model 1 and model 2 lattices. DOS, Density of state.

consideration of the EMU model [22,28] needs to take the nonequivalence of Gd/Lu ions in model 2 lattice into account, in order to approach higher efficiency of ET between inter-RE doping ions. We deduced that β-NaLnF4 (Ln 5 Y, Gd, and Lu) are basically charge transfer solids that have nearly the same band gaps, regardless of the local occupational disorder of Na or Y, as well as the Gd. The only slight difference is the local coordinated F sites. The lattice with an F site with fivefold coordination has energy drop due to a strong charge transfer between F and cation sites near the Γ point of the first BZ. In contrast, the lattice with an F site with only fourfold coordination has energy maximum at the Γ point. The VB DOS shows sharp peaks denoting the energy surface given by different F sites around the first BZ which is nearly flat. We here deduce that more than one excitation state has participated in the EMU process crossover the interface with relative support from previous experiments. Qin et al. have studied the 4f fine levels of Gd31 and discussed the three different excitation ranges: 6P7/2, 6IJ, and 6DJ [73]. There has been also a twin-peak profile found below 270 nm in the emission spectrum for 6IJ, denoting a thermal transition. Moreover, Qin et al. also found that the ET is

Chapter 20 • Electronic Structures of Alkaline Rare Earth

461

FIGURE 20–10 Schematic diagram of the interface and band offsets models of an α-NaYF4@SiO2 heterogeneous core
shell system (α-NaYF4@SiO2) illustrated with VBO and CBO [76]. CBO, Conduction band offset; VBO, valence band offset.

rather efficient from 3P2 of Tm31 to 6DJ of Gd31 instead of 6P7/2 of Gd31, with a quantum yield of 0.78 [73]. Liu et al. have shown that Gd31 can efficiently migrate through a resonant quadrupole
quadrupole ET to transport the photons to the activators like Dy31 and Eu31 in NaGdF4 NCs, from 6IJ (Gd31)!6PJ (Gd31)!6P7/2 (Dy31) and 6IJ (Gd31)!6PJ (Gd31)!5D4 (Eu31), respectively [74]. Podhorodecki et al. have shown a similar diagram for presenting a possible route to migrate the energy from Gd31 to Eu31 activators, and three different ranges near the 300 nm of excited states Gd31 have been taken into account for this ET process [75]. As an outlook part to this chapter, we blueprint future interface band offset engineering as a tunable physical variable on the RE Ln core
shell nanostructured materials (Fig. 20
10). This is to achieve the future engineering of UC luminescence and possible extensions to other Ln nanomaterials. Given our systematically established DFT methodology development at the preliminary stage, we can apply the excited-state theory, including LR-TDDFT and LR-TDDFT 1 U methods, to study the electronic state properties (such as excitation energy and oscillator strength) and excitation spectrum of the Ln core
shell nanomaterials, and the dynamics of excited-state physicochemical trend. The outlook research will be on the interface band offset with the choice of various Ln ionic crystals, in energy, optical devices, and in vivo bio-imaging applications. When studying the ground-state properties of core
shell interfaced nanomaterials, we may further, in theory, achieve the following databases for experimental reference: 1. Accurate and reliable electronic structures and optical properties; 2. The intrinsic single-particle and optical vertical excited-transition levels; 3. Extension of the computational method for interface band offset engineering; 4. Inversed strategy for band structure for materials design.

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THERANOSTIC BIONANOMATERIALS

Note that, in previous theoretical development [45,77], we systematically demonstrated that the challenge could be ascribed to the incomplete cancellation of electron
electron Coulomb repulsion energy under external perturbation. We applied the secondary charge response, denoted the “screened pseudo-charge potential” model, to offset such residue effects. Counteracting between these two charge-response-induced Coulomb potentials, the U parameters are self-consistently obtained by fulfilling the conditions for minimizing the non-Koopmans energy. Referring to the work of Kulik et al. [78], we can easily rewrite the output U parameter based on the orbital potential component terms as Uout1 and Uout2 generated from our developed method, given as [45,77]: 




@αI @αKS @αa @αKS Uin Uin Uin I a 2 2 5 2 U 5 2 U Uout1 5 2 ð U Þ 2 scf1 in @qðaÞ @qðaÞKS @qðaÞ @qðaÞKS a0 a0 m
 @αI @αKS I Uout2 5 2 2 @qðbÞ @qðbÞKS

(20.1)

(20.2)

When the two Hubbard parameter outputs induced by different charge response systems become equivalent, Uscf is thus determined by an input Uin when the two output U values have Uout2 2 Uout1 5 0. Therefore the relationship between Uscf and Uin can be described by h  Uin Uscf 5

m

h

2

 

11

a0 Uin



a0 Uin

@Uin @qI

i

i

(20.3)

Through treating localized orbitals, we have successfully unified the hybrid-DFT and DFT 1 Uscf by following Ivady and Gali et al. [45,77,79,80], in which they justifiably added a DFT 1 U-like on-site potential correction in hybrid-DFT. The additional total energy term is ΔEXPBE0

 

 σ  αHF F 0 2 J 0 X h σ  σ 2 i nm 5 nm 2 nm 2 m;σ

(20.4)

Therefore the 4f
4f Coulomb repulsion energy is self-consistently described by our method shown above. This pseudocharge potential model within the corrected linearresponse framework is flexible enough to solve the on-site effective screened Coulomb potential for cations and anions in solids with either fully (closed shell) or partially occupied (open shell) orbitals. Since this method can quickly give electronic structures of the eigenbulk properties but also provide satisfactory native defect levels of bulk or low-dimensional structures, it can help propel our research and guide the discovery of next-generation highthroughput electronic engineering materials, which are formed or synthesized under extreme physical or chemical environments, at a faster pace. Thus our model is suitable to deal with large-scale solid material in considering both accuracy and efficiency of calculations. From our preliminary work [76], we have confirmed the intrinsic efficiency of the Förster resonance energy transfer (FRET) from the host matrix correlated with the interface band

Chapter 20 • Electronic Structures of Alkaline Rare Earth

463

offset, shell-to-core thickness ratio, and interface bonding energy in area density, which is described as follows:   FRET PIF2dipole ~16π2 ε0 R 2Rt 2 R2 jσS jðΔVBO Þ2  32π2 R2 ε0 t jσS jðΔVBO Þ2 ;

(20.5)

where σS represents the interface energy area density, t is the interlayer distance, and ΔVBO is the interface band offset. This only represents the contribution from the host lattice of the interface region between the core and shell to the ET. The nonradiative ET approximated with the FRET model for core
shell is roughly updated to be: 0

FRET LRET Pcore
shell 5 PSA  PSA 1 PIF2dipole 1 PIF2IBIGS ;

(20.6)

LRET where the third term, PIF2IBIGS is the ET probability in radiative form as luminescence resonance ET, which has a distance dependence of R22 decaying behavior, and is also deterÐ mined by the spectra overlap of ge ðvÞgh ðAÞdv similarly. Previously, Dorenbos comprehensively illustrated the significance of the relative positions of Ln levels within the optical band gap. We formulated this into the 4f vacuum referred binding energy (4f-VRBE) levels [47,48,55]. With our first-principles two-way crossover linear response method (described above), the 4f
4f Coulomb repulsion potential energy of Ln dopants can be self-consistently predicted [45,77], which is demonstrated in terms of U (Ln, vacuum), where the vacuum denotes the vacuum level. Further, using this Hubbard-U parameter within the DFT 1 U framework, the Ln dopant binding energy relative to the host matrix can be determined, as shown in Fig. 20
11. Through the 4f-VRBE levels, accurate estimations on the lowest 4f-excited states of the whole Ln dopant ions (Ln 5 La. . .Lu) within the host either core or hole will be consequently achieved as:

U ðn; AÞ  E4f ðn 1 1; 2 1 ; AÞ 2 E4f ðn; 3 1 ; AÞ

(20.7)

E4f ðm; Q; AÞ 5 E4f ðm; Q; vacuumÞ 1 E ðEu; Q; AÞ 1 αðQ; AÞRðmÞ

(20.8)

The simulation of dynamics processes involved in multiphoton excitations and deexcitations of interfacial core
shell nanomaterial are still missing from the literature, despite the fact that their potential-energy surfaces and configurations of electronic ground and excited states have been obtained using various excited state methods. It is necessary to solve the nonadiabatic coupling term [81
83] or the nonadiabatic coupling vector. With our

developed method, the ground-state wavefunction ψðrÞ has been accurately described. By implementing the time-dependent expansion coefficients, e.g., nuclear trajectory R(t), the

P

excited state wavefunction updates to ψðr; RðtÞÞ 5 K CK ðtÞ ψK ðr; RðtÞÞ , following Tully’s surface hopping method [81
83]. Overall, the electronic wave function still maintains the

basis of adiabatic Born
Oppenheimer states. Such excited state wavefunction ψK ðr; RðtÞÞ , represents the adiabatic electronic state K, while CK(t) is the time-dependent expansion coefficients. In addition, the nuclei trajectories R(t) can be calculated by the classical Newton’s

464

THERANOSTIC BIONANOMATERIALS

FIGURE 20–11 (A) Band offsets of different lanthanide ionic crystal materials. (B) Schematic core
shell structures. (C) Preliminary data on the relative lanthanide dopant ion-level positions within the optical band gap of the β-NaGdF4 host.

equations of motion. The time evolution of the coefficients CK(t) along a given trajectory can be obtained by solving the time-dependent Schrödinger equation: ih ¯

 X @ψ ðr; RðtÞÞ dCK ðtÞ 5 EK CK ðtÞ 2 ih ¯ ψK ðr; RðtÞÞ I CI ðtÞ dt @t I

(20.9)

where EK is the energy for the state K. For the future development of TDDFT 1 U within a linear response method, combination with mean-field QM/MM method is necessary. Nanostructured models for Ln-based luminescence fluorides will be constructed to investigate the surface quenching effect of both the interface between the core and shell, and the shell surface region on their influence to the ET. To connect our DFT models with the actual UCNPs, the constructed models must be more like the real core
shell structure rather than a pure crystal lattice. For the core part in a core
shell system, the model is realized by the slab on the conventional crystal lattice to a sphere morphology through our own developed codes. As the size of the system enlarges, the sphere morphology and surface effect induced by more dangling bonds and isolated ions should be more evident. The shell model originates by removing a sphere from the already-cut core models, in which the thickness of the shell can be modified through coding control. By altering the thickness, we can illustrate the thickness effect from the shell to the luminescence properties. Since many combinations of core and shell molecules have been synthesized, we will build a series of core
shell structural models based on combinations from different luminescence materials to uncover the effect from the interaction between materials to ET and the band offset from homogeneous and heterogeneous interface effects. As well as the core
shell structure, we are also still

Chapter 20 • Electronic Structures of Alkaline Rare Earth

465

working on novel NPs based on our developed slab method. Recently, a new tube structure model has been constructed with the modulated thickness, which has potential application in drug delivery or bioimaging. With these special nanostructure models, geometry relaxation, electronic structure calculation, and excitation state confirmation will be conducted under the DFT 1 U method. Hence, we can clearly observe the bonding variation, surface rearrangement, and interface change with or without dopants that are all significant in revealing the mechanism of UC luminescence.

20.7 Summary In summary, we have investigated the electronic structures β-NaLnF4 (Ln 5 Y, Gd, and Lu) in P-6 symmetry of β-phase. The atomic structure correlations to the band structures and electronic properties have also been discussed. We found the p-d optical transition consists of the band gap of β-lattice, but relative positions of 4f levels in the gap result in different observed band gaps. We discussed that the local disorder of fluoride modulates the electronic eigenvalues of the top of VB near the Γ point within the first BZ. The CB minimum is always located at the Γ point consisting of the d orbitals of Y/Gd/Lu, regardless of siteoccupation disorder between Y/Gd/Lu and Na. Based on this work, we proposed a convenient route for future investigation of the interface states that potentially quench the UC luminescence.

Acknowledgments The author gratefully acknowledges the support of the Natural Science Foundation of China (NSFC) for the Youth Scientist grant (Grant No. NSFC 11504309, 21771156), the initial start-up grant support from the Department General Research Fund (Dept. GRF) from ABCT in the Hong Kong Polytechnic University, and the Early Career Scheme (ECS) Fund (Grant No. PolyU 253026/16P) from the Research Grant Council (RGC) in Hong Kong.

References [1] H.-X. Mai, Y.-W. Zhang, L.-D. Sun, C.-H. Yan, J. Phys. Chem. C 111 (2007) 13721. [2] L.-D. Sun, Y.-F. Wang, C.-H. Yan, Acc. Chem. Res. 47 (2014) 1001. [3] N. Bogdan, F. Vetrone, G.A. Ozin, J.A. Capobianco, Nano Lett. 11 (2011) 835. [4] A. Sedlmeier, H.H. Gorris, Chem. Soc. Rev. 44 (2015) 1526. [5] G. Chen, H. Agren, T.Y. Ohulchanskyy, P.N. Prasad, Chem. Soc. Rev. 44 (2015) 1680. [6] G. Chen, H. Qiu, P.N. Prasad, X. Chen, Chem. Rev. 114 (2014) 5161. [7] A. Gnach, T. Lipinski, A. Bednarkiewicz, J. Rybka, J.A. Capobianco, Chem. Soc. Rev. 44 (2015) 1561. [8] A.M. Ibarra-Ruiz, D.C. Rodríguez Burbano, J.A. Capobianco, Adv. Phys. X 1 (2016) 194. [9] Y.I. Park, K.T. Lee, Y.D. Suh, T. Hyeon, Chem. Soc. Rev. 44 (2015) 1302. [10] W. Zheng, P. Huang, D. Tu, E. Ma, H. Zhu, X. Chen, Chem. Soc. Rev. 44 (2015) 1379.

466

THERANOSTIC BIONANOMATERIALS

[11] Y. Sun, W. Feng, P. Yang, C. Huang, F. Li, Chem. Soc. Rev. 44 (2015) 1509. [12] M.-K. Tsang, G. Bai, J. Hao, Chem. Soc. Rev. 44 (2015) 1585. [13] E.M. Chan, Chem. Soc. Rev. 44 (2015) 1653. [14] B. Zhou, B. Shi, D. Jin, X. Liu, Nat Nanotechnol. 10 (2015) 924. [15] X. Chen, D. Peng, Q. Ju, F. Wang, Chem. Soc. Rev. 44 (2015) 1318. [16] K.A. Abel, J.-C. Boyer, F.C.J.M. van Veggel, J. Am. Chem. Soc. 131 (2009) 14644. [17] K.A. Abel, J.-C. Boyer, C.M. Andrei, F.C.J.M. van Veggel, J. Phys. Chem. Lett. 2 (2011) 185. [18] H. Dong, L.-D. Sun, C.-H. Yan, Chem. Soc. Rev. 44 (2015) 1608. [19] F. Wang, J. Wang, X. Liu, Angew. Chem. Int. Ed. 49 (2010) 7456. [20] F. Wang, X. Liu, Acc. Chem. Res. 47 (2014) 1378. [21] R. Deng, F. Qin, R. Chen, W. Huang, M. Hong, X. Liu, Nat Nanotechnol. 10 (2015) 237. [22] F. Wang, R. Deng, J. Wang, Q. Wang, Y. Han, H. Zhu, et al., Nat. Mater. 10 (2011) 968. [23] X. Liu, R. Deng, Y. Zhang, Y. Wang, H. Chang, L. Huang, et al., Chem. Soc. Rev. 44 (2015) 1479. [24] X. Li, F. Zhang, D. Zhao, Chem. Soc. Rev. 44 (2015) 1346. [25] Y.-F. Wang, L.-D. Sun, J.-W. Xiao, W. Feng, J.-C. Zhou, J. Shen, et al., Chem. Eur. J. 18 (2012) 5558. [26] F. Zhang, R. Che, X. Li, C. Yao, J. Yang, D. Shen, et al., Nano Lett. 12 (2012) 2852. [27] X. Li, D. Shen, J. Yang, C. Yao, R. Che, F. Zhang, et al., Chem. Mater. 25 (2013) 106. [28] Q. Su, et al., J. Am. Chem. Soc. 134 (2012) 20849. [29] N.J.J. Johnson, A. Korinek, C. Dong, F.C.J.M. van Veggel, J. Am. Chem. Soc. 134 (2012) 11068. [30] K. Prorok, A. Bednarkiewicz, B. Cichy, A. Gnach, M. Misiak, M. Sobczyk, et al., Nanoscale 6 (2014) 1855. [31] S. Han, X. Qin, Z. An, Y. Zhu, L. Liang, Y. Han, et al., Nat. Commun. 7 (2016) 13059. [32] X. Chen, L. Jin, W. Kong, T. Sun, W. Zhang, X. Liu, et al., Nat. Commun. 7 (2016) 10304. [33] B. Chen, D. Peng, X. Chen, X. Qiao, X. Fan, F. Wang, Angew. Chem. Int. Ed. 54 (2015) 12788. [34] B. Zhou, L. Tao, Y. Chai, S.P. Lau, Q. Zhang, Y.H. Tsang, Angew. Chem. Int. Ed. 55 (2016) 12356. [35] R.T. Tung, Phys. Rev. Lett. 84 (2000) 6078. [36] R.T. Tung, Phys. Rev. B 64 (2001) 205310. [37] J. Robertson, J. Vac. Sci. Technol. B 18 (2000) 1785. [38] J. Robertson, Eur. Phys. J. Appl. Phys. 28 (2004) 265. [39] F. Auzel, . in in: B. DiBartolo, V. Goldberg (Eds.), Radiationless Processes, Springer, Boston, MA, 1980, p. 213. [40] F. Auzel, Chem. Rev. 104 (2004) 139. [41] T. Förster, Ann. Phys. 437 (1948) 55. [42] D.L. Dexter, J. Chem. Phys. 21 (1953) 836. [43] P.W. Anderson, Phys. Rev. 109 (1958) 1492. [44] C. Huang, Z. Bian, in Rare Earth Coordination Chemistry, John Wiley & Sons, Ltd, 2010p. 1. [45] B. Huang, J. Comput. Chem. 37 (2016) 825. [46] J.W. Stouwdam, M. Raudsepp, F.C.J.M. van Veggel, Langmuir 21 (2005) 7003. [47] P. Dorenbos, J. Lumin. 108 (2004) 301. [48] P. Dorenbos, Chem. Mater. 17 (2005) 6452.

Chapter 20 • Electronic Structures of Alkaline Rare Earth

467

[49] X.Y. Chen, H.Z. Zhuang, G.K. Liu, S. Li, R.S. Niedbala, J. Appl. Phys. 94 (2003) 5559. [50] J. Wang, et al., Nat. Mater. 13 (2014) 157. [51] G. Yao, M.T. Berry, P.S. May, D.S. Kilin, Int. J. Quantum. Chem. 112 (2012) 3889. [52] G. Yao, M.T. Berry, P.S. May, D. Kilin, J. Phys. Chem. C 117 (2013) 17177. [53] G. Yao, S. Huang, M.T. Berry, P.S. May, D.S. Kilin, Mol. Phys. 112 (2014) 546. [54] K. Chong, T. Hirai, T. Kawai, S. Hashimoto, N. Ohno, J. Lumin. 122
123 (2007) 149. [55] P. Dorenbos, Phys. Rev. B 85 (2012) 165107. [56] A. Zunger, A.J. Freeman, Phys. Rev. B 16 (1977) 2901. [57] J.F. Janak, Phys. Rev. B 18 (1978) 7165. [58] C.-C. Lee, H.C. Hsueh, W. Ku, Phys. Rev. B 82 (2010) 081106. [59] C.J. Pickard, B. Winkler, R.K. Chen, M.C. Payne, M.H. Lee, J.S. Lin, et al., Phys. Rev. Lett. 85 (2000) 5122. [60] T. Zacherle, A. Schriever, R.A. De Souza, M. Martin, Phys. Rev. B 87 (2013) 134104. [61] P.R.L. Keating, D.O. Scanlon, B.J. Morgan, N.M. Galea, G.W. Watson, J. Phys. Chem. C 116 (2011) 2443. [62] B. Huang, R. Gillen, J. Robertson, J. Phys. Chem. C 118 (2014) 24248. [63] B. Huang, Philos. Mag. 94 (2014) 3052. [64] S.J. Clark, M.D. Segall, C.J. Pickard, P.J. Hasnip, M.I.J. Probert, K. Refson, et al., Z. Kristallogr. 220 (2005) 567. [65] L. Kleinman, D.M. Bylander, Phys. Rev. Lett. 48 (1982) 1425. [66] S.G. Louie, S. Froyen, M.L. Cohen, Phys. Rev. B 26 (1982) 1738. [67] I. Grinberg, N.J. Ramer, A.M. Rappe, Phys. Rev. B 62 (2000) 2311. [68] A.M. Rappe, K.M. Rabe, E. Kaxiras, J.D. Joannopoulos, Phys. Rev. B 41 (1990) 1227. [69] I.A. Vladimir, F. Aryasetiawan, A.I. Lichtenstein, J. Phys. Condens. Matter. 9 (1997) 767. [70] N. Marzari, D. Vanderbilt, M.C. Payne, Phys. Rev. Lett. 79 (1997) 1337. [71] B. Huang, Solid State Commun. 230 (2016) 49. [72] B. Huang, Inorg. Chem. 54 (2015) 11423. [73] W. Qin, et al., Opt. Lett. 33 (2008) 2167. [74] H. Guan, G. Liu, J. Wang, X. Dong, W. Yu, New J. Chem. 38 (2014) 4901. [75] M. Banski, A. Podhorodecki, J. Misiewicz, M. Afzaal, A.L. Abdelhady, P. O'Brien, J. Mater. Chem. C 1 (2013) 801. [76] B. Huang, H. Dong, K.-L. Wong, L. Sun, C. Yan, J. Rare Earths 35 (2017) 315. [77] B. Huang, Phys. Chem. Chem. Phys. 19 (2017) 8008. [78] H.J. Kulik, M. Cococcioni, D.A. Scherlis, N. Marzari, Phys. Rev. Lett. 97 (2006) 103001. [79] V. Ivády, I.A. Abrikosov, E. Janzén, A. Gali, Phys. Rev. B 87 (2013) 205201. [80] V. Ivády, R. Armiento, K. Szász, E. Janzén, A. Gali, I.A. Abrikosov, Phys. Rev. B 90 (2014) 035146. [81] E. Tapavicza, I. Tavernelli, U. Rothlisberger, Phys. Rev. Lett. 98 (2007) 023001. [82] I. Tavernelli, E. Tapavicza, U. Rothlisberger, J. Chem. Phys. 130 (2009) 124107. [83] R. Mitri´c, U. Werner, M. Wohlgemuth, G. Seifert, V. Bonaˇci´c-Koutecký, J. Phys. Chem. A 113 (2009) 12700.

21 Lanthanide-Based Magnetic Resonance Imaging MetalResponsive Agent Overview Lei Zhang, Kai Liu STAT E K EY LABORATORY OF RARE EARTH R ESOURCE UTILIZATION, CHANGCHUN INSTITUTE OF APPLIED CHEMISTRY, CHINESE A CADEMY OF SCIENCES, C HANGCHUN, P.R. CHINA

21.1 Introduction Magnetic resonance imaging (MRI) has become a valuable imaging facility owing to its excellent spatial and anatomical resolution over the past 30 years. In particular its nonradiation property has made it more attractive than other imaging modalities such as X-ray, computed tomography (CT), positron-emission tomography (PET), and single-photon emission computed tomography (SPECT) [1]. The signal generated from MRI is the relaxation of a transverse component of net magnetization of protons in the bulk water that is present in the body [2]. Lanthanide-based small-molecule MRI contrast agent has attracted more attention over the last 20 years and its important roles when considering the possibility for cancer and other disease diagnoses during MRI examination is expanding. Typically, lanthanide-based MRI contrast agents can be divided into three groups through the water exchange rate between bound and bulk water: T1 (fast water exchange rate); T2ex (slow to intermediate exchange rate); and chemical exchange saturation transfer (CEST) (slow water exchange rate) based agents. Among these, gadolinium-based MRI contrast agent has been thoroughly investigated and made tremendous improvements from basic theory to clinical applications. In addition, T2ex and paramagnetic CEST (paraCEST) agents are still in the stage of basic chemistry with few preclinical applications. T1 and T2 MRI contrast agents, they accelerate nuclear relaxation mainly through a dipole interaction, significantly enhancing the contrast of images. One of the inspiring frontiers of MRI contrast agents is the development of responsive probes, which can be used to report chemical species in biological systems and the chemical reactions of interest [3,4]. A responsive MRI probe could provide a signal selectively, depending on the specific biological or physiological parameter [5]. In this chapter, we first discuss the basic theories on gadolinium contrast agents and the relaxivity change mechanism, and paraCEST agents’ intensity change mechanism, followed by recent progress on T1 and paraCEST-type metal-responsive contrast agents. Instead of Theranostic Bionanomaterials. DOI: https://doi.org/10.1016/B978-0-12-815341-3.00021-3 © 2019 Elsevier Inc. All rights reserved.

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presenting a comprehensive review of responsive agents from small molecules in biological environment that are related to disease, we focus on the most recent research work related to gadolinium and paraCEST-based MRI contrast agents. Finally, we close with a discussion on the future direction of exploration and perspectives on these three subgroups of the agent.

21.2 Gadolinium Magnetic Resonance Imaging-Responsive Agents 21.2.1 The Relaxivity Change Mechanism Gadolinium ions, containing seven unpaired electrons in 4f orbital, have the ability to decrease both the longitudinal and transverse relaxation times (T1 and T2) by relaxation of nearby nuclei [6,7]. In order to avoid toxicity, gadolinium ions must be chelated by multidentate ligands, especially the macrocyclic ligands, due to their thermodynamic stability which can form a stable complex that can remain intact through the imaging process or even later [8,9]. Normally gadolinium complexes have at least one water molecule that coordinates with a metal ion which plays the most important role through the relaxation process by exchange with the bulk water proton. In turn, the relaxation effect would finally transfer to the bulk water which induces the decrease on T1. The efficiency of gadolinium contrast agents is related to its relaxivity and is presented in Eq. (21.1). In the equation, T1 refers to the relaxation time, T1O refers to the relaxation time of contrast agent free solution, r1 refers to the relaxivity of the contrast agent, and [CA] is the concentration of the contrast agent. 1 1 5 1 r 1 ½CA Τ1 Τ1O

(21.1)

There are many factors that can lead to a change in the relaxation rates of macrocyclicbased gadolinium complexes, as shown in Fig. 211. Solomon's Bergen Morgan equation, which has been very well explained elsewhere, is often used to describe the relationship between variables leading to inner relaxation rates [10,11]. Herein, three important factors are mainly discussed, including the number of inner-sphere water molecules (q), the rotational tumbling time (τ R), and the residence lifetime of inner-sphere water molecules (τ m). The longitudinal relaxivity is proportional to the number of inner-sphere water molecules. Therefore the modulation of the inner-sphere number of bound water molecules would change the relaxivity in terms of a certain response analyte. A higher number of bound water molecules would increase its relaxivity while it would decrease the thermodynamic stability of the complex. A longer rotational tumbling time always leads to higher relaxivity and this has been fully proved by several gadolinium complexes that bind to bovine serum albumin and human serum albumin (HSA), and the longitudinal relaxivity would be increased by almost one level of magnitude. Furthermore, the water residence lifetime has been evidenced by the theory to have an optimal value in terms of longitudinal relaxivity in the range

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FIGURE 21–1 Factors that affect the longitudinal relaxivity.

of 10281027 s. Most prevalently, the preclinically and clinically approved contrast agents have a too slow water exchange rate. Through the chemical method it is difficult to improve the relaxivity for the following reasons. If the exchange rate is too fast, r1 is limited since the time that the water molecule is bound to the metal ion is not long enough to be completely relaxed. In contrast, if the exchange rate is too slow, r1 is also limited due to fewer water molecules being bound to the paramagnetic center hindering transfer of the maximum effect to the bulk water’s T1 [8,12].

21.2.2 Survey of Gd-Based Magnetic Resonance Imaging Sensors 21.2.2.1 Modulation of the Inner Sphere Number of Water Molecules Calcium has been considered one of the most important elements in the living system and it usually exists in the form of calcium ions. In the human body, most calcium ions are stored in the teeth and skeleton, and very few are present in blood tissue. There is a scale of micromolar calcium ions inside cells and millimolar concentrations outside cells. Also, it has been considered as the second messenger in cell signaling processes throughout the body [13]. Several types of calcium MRI-responsive agents are illustrated in Fig. 212. Meade et al. designed the first Ca21-responsive agent, DOPTA-Gd (Gd-1). Two DO3A derivatives were connected together by 1,21,2-bis(o-aminophenoxy)ethane-N,N,N0N0-tetraacetic acid moiety. This functional group, which has been approved, could bind with Ca ions in relatively high

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FIGURE 21–2 Calcium (Gd-1 and Gd-2) and copper (Gd-3 and Gd-4)-activated MRI contrast agents. MRI, Magnetic resonance imaging.

binding affinity. In the absence of Ca21, both acetate groups on the iminodiacetate would coordinate with Gd ions and the total relaxivity of the contrast agent is then 3.26 mM21 s21. In the presence of one equivalence of Ca21, the relaxivity would be increased by 80% to 5.76 mM21 s21 due to the acetate groups on the iminoacetate having better binding affinity toward the calcium ion and this would leave two spaces for the bound water molecules to coordinate to the Gd ion (q 5 01.5). Also, the better selectivity and higher binding affinity of this contrast agent to calcium ions has been further approved [14]. Subsequently, a complex containing only one Gd-DO3A derivative Ca sensor has been discussed recently, as shown in Fig. 212 (Gd-2) [15]. Upon adding one equivalence of Ca21, the relaxivity was increased from 3.5 to 6.9 mM21 s21. The binding affinity of this agent to Ca ions is in the micromolar range. Thus this agent also has affinity to Mg21 and Zn21 and the relaxivity would be increased with either. In addition, this calcium sensor was also enclosed in a study of artificial cerebrospinal fluid and artificial extracellular matrix in 37 simulated biological conditions and showed 36% and 25% relaxation enhancement, respectively, in response to Ca21. Copper is also an indispensable trace element in living systems, including in humans, and is crucial for many organ and metabolic processes. An excess or deficiency of copper in the human body can induce several diseases, including Menkes’, Wilson’s, and Alzheimer’s diseases [16]. In recent years, many efforts have been made to develop copper sensors, including on MRI, fluorescence, and PET imaging facilities. Using MRI there are two typical strategies on designing gadolinium-based copper sensors that are broadly applied in chemistry laboratories. Chang et al. developed the first generation of Gd-DO3A derivative copper sensors presenting a 41% increase on relaxivity from 3.76 to 5.29 mM21 s21 with the presence of 1 equivalence of copper ion, with the structure shown by Gd-3 [17]. Also, it exhibits very high binding affinity that is in the micromolar range (Kd 5 167 μM). In addition, this copper sensor shows excellent copper selectivity compared with other important metal ions

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in the PBS and HEPES buffer. The responsive mechanism was due to the iminodiacetate having better affinity with copper compared with Gd ions. On this occasion, the q of Gd ions would be increased to 2, which led to an enhancement of the relaxivity. Another interesting method to image copper is the bimodal method using MRI and an optical imaging modality, as shown in Fig. 212 (Gd-4) [18]. This sensor was a combination of the fluorescence group and Gd-DPTA derivative. In the presence of one equivalence of copper, the fluorescence of the agent was quenched due to the paramagnetic nature of the Cu21 ions and the relaxivity was increased from 2.01 to 4.01 mM21 s21. Also, this agent is very selective to copper over other biological relevant metal ions in both fluorescence and relaxivity studies. Moreover, the agent shows no cell toxicity and has the ability to image copper by fluorescence in RAW 264.7 cell lines. This bimodal method may find preclinical and clinical applications, especially in intraoperative procedures, and MRI could be used to localize diseased tissues and the optical modality could be used in biopsies for histological validation.

21.2.2.2 Modulation of Rotational Tumbling Time With an increase in rotational tumbling time of the contrast agent, the longitudinal relaxivity is known to also increase. For example, MS-325 could selectively bind to HSA [1]. As a consequence, the relaxivity shows the enhancement from 6.84 to 15.2 mM21 s21, which would induce better contrast behavior in terms of in vivo applications [19]. In recent years, divalent zinc has been demonstrated to play a critical role in many biological metabolic processes. The total concentration of zinc ions in the blood can reach 16 μM, which mostly has been chelated or bound to protein [2022]. Pancreatic beta cells store insulin with two equivalences of zinc and release free zinc in response to an increase concentration in plasma glucose. Zn(II) is tightly regulated by multiple different transporters, and imbalances in Zn(II) content in these various tissues is associated with diabetes, Alzheimer’s disease, and prostate cancer [23]. This triggers most of research in the field which has started to pay more attention to the importance of zinc. Many types of zinc-responsive agents have been designed and discussed. Fig. 213 presents several zinc-responsive agents. Mostly, the Gd-DOTA derivatives contain N,N-bis (2-pyridyl-methyl)ethylenediamine (BPEN), which shows high binding affinity functional group to zinc ions on its macrocyclic ring. In the presence of two equivalences of zinc ions, the relaxivity of Gd-3 increases from 5.0 to 6.6 mM21 s21. Most interestingly, after binding with HSA, the relaxivity would be further increased to 17.4 mM21 s21 [24]. More works have been performed by tuning the water exchange rate through chemical methods to increase the relaxivity after binding with HSA. Sherry and co-workers have reported the highest relaxivity of zinc sensors as 50.1 mM21 s21 after binding with HSA [25]. The reason for this is that one of the side arm chains has one extra methylene carbon inserted into one ligating sidechain, which increases substantially the water exchange rates of the complex. Several similar structures have been constructed based on the contribution of the side arm chain effect to the water exchange rate and give relatively higher relaxivity compared with Gd-5. It has been considered that a significant change in the relaxivity for the complex-zinc binding to HSA is a result of a longer τ R value. The corresponding sensors were successfully applied in vivo for

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FIGURE 21–3 Zinc-activated MRI contrast agents. MRI, Magnetic resonance imaging.

zinc imaging in prostate cancer-related disease. Through this method for detecting zinc, there is great potential to detect a change of free zinc ion concentration in biological metabolism processes. Nevertheless, from a chemistry point of view, the experimental relaxivity of the contrast agent is still far from the theoretical value due to τ R not being very well optimized. The next generation of zinc sensor should have higher binding affinity toward HSA, which in turn would increase τ R.

21.3 Survey of the Paramagnetic Chemical Exchange Saturation Transfer Responsive Agent paraCEST is a relatively new MRI technique that has been investigated in the MR field originating from magnetization transfer (MT), which is a common physical phenomenon described by Forsen and Hoffman in 1963 [26]. The mechanism of CEST is based on the detection of different pools of nuclear spins, separated by different chemical shifts, in chemical exchange with one another. The first CEST agent was reported by Balaban in 2000 using this technique to produce contrast in NMR [27]. In the following year, a paraCEST agent was developed by Sherry et al. [28]. Thus CEST could be explored with diamagnetic and paramagnetic molecules (diaCEST and paraCEST). In biological media, a CEST agent requires labile protons to exchange with bulk water protons (Fig. 214). Furthermore, the exchange with water can also be modulated. Typically, a moderate to slow exchange rate (kex) is

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FIGURE 21–4 Schematic representation of the distribution of spins and the simulated NMR spectra when a presaturation pulse is applied and the system is undergoing chemical exchange.

preferable for CEST [8]. The kex is not the only factor governing CEST; in fact, both kex and Δω are linked in CEST [Eq. (21.2)]. kex # Δω

(21.2)

In order to produce a signal through a CEST mechanism, the kex must be smaller than the chemical shift difference (Δω). When this condition is satisfied and the labile pool of protons is detected, one can saturate it by applying a selective radiofrequency pulse. After the pulse saturation, the labile pool of protons exchanges spins with the bulk water, and transfer is observed as a decrease in the bulk water CEST signal to yield negative contrast. One of the advantages of CEST agents compared to traditional T1 and T2 contrast agents is that the tissue contrast can be turned “on” and “off” by applying a selective radiofrequency pulse [29]. diaCEST agents normally are of low molecular weight and contain endogenous diamagnetic molecules, which contain amide, hydroxyl, or amine groups. This presents a smaller chemical shift difference between labile protons and solvating water protons, exhibiting an optimal slower kex. Barbituric acid, shown in Fig. 215, was the first example of a diaCEST agent. By applying saturation power on an exchangeable amide proton

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FIGURE 21–5 Chemical structure of barbituric acid as the first diaCEST agent in MRI and CEST spectra of 125 mM (blue), 62.5 mM (red), and 31.25 mM (green) solutions of barbituric acid recorded at 300 MHz, pH 7.0, and 37 C. CEST, Chemical exchange saturation transfer; diaCEST, diamagnetic chemical exchange saturation transfer; MRI, magnetic resonance imaging.

(5 ppm compared to the water proton signal), the bulk water signal decreased by 32% at a concentration of 68 mM. However, the small Δω makes it hard to perform selective saturation without interfering with the bulk water signal. Another challenge is that there are numerous other exchangeable protons present in vivo in the diamagnetic NMR chemical shift range. Given the challenges of the diaCEST agent, another type of CEST modality introduced paraCEST agents. Typically, paraCEST agents contain a highly shifted bound water molecule coordinated to an Ln metal ion and produce CEST signals with a much larger and faster exchange rate [1]. The first paraCEST agent reported in the literature was an Eu-based macrocyclic complex: EuDOTA(gly)4 [30]. The chemical structure of this Eu complex is similar to GdDOTA, but with amides in place of the carboxylate groups found in GdDOTA, yielding a slower water exchange rate for the bound water. EuDOTA(gly)4 has been widely studied in the past for its detectable bound water pool of protons, shifted to 50 ppm at 298K with moderate-to-slow proton exchange rate [28]. The first advantage of paraCEST over diaCEST agents is the large chemical shifts of exchangeable protons that allow for direct saturation at specific frequencies without interfering with the bulk water. Another advantage is the better shifting ability of some lanthanide metals, yielding a further shifted bound water molecule signal. This prevents the MT effect caused by the semisolid nature of biological tissues with a very long T2

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value [31]. A third advantage is that, by altering the side arm, the complex could be designed as an agent responsive to several biological factors, such as pH, temperature, and the presence of metallic ions and enzymes. However, only a few CEST agents that are capable of being responsive to metal ions have been studied to date.

21.3.1 Zinc and Calcium Chemical Exchange Saturation Transfer Responsive Agent As already mentioned about the importance of zinc and calcium ions in disease-related processes, research in the field of CEST agents also has attracted more attention for zinc ion imaging. Sherry et al. have design a DOTAM type agent bearing BPEN on the set arm chain on its macrocyclic ring showing high binding affinity functional group to zinc ions (Fig. 216). In the presence of one equivalence of zinc ions at pH 7, the CEST signal intensity was decreased, probably due to the faster water exchange once the zinc ions bind to the pyridine group and a similar result was obtained when the pH was increased to 8. The reason for this is probably due to the additional zinc ion itself having a coordinated water molecule that is partially deprotonated at these pH values, thus positioning a zinc OH species near the EuIII-bound water molecule. This species could act to catalyze prototropic exchange between the EuIII-bound water molecule and the bulk water proton. Another interesting phenomenon in this research work was that only one zinc ion could bind to one Eu complex under all experimental conditions, which presumably indicates that one zinc ion would coordinate with four pyridine groups while leaving the tertiary amine on the side arm only [32]. Angelovski et al. designed two calcium-responsive CEST agents which used Eu/Yb-DO4A (gly)4 derivatives as platform (Fig. 217). Due to the faster water exchange rate properties of Yb-based macrocyclic complex, they detected the CEST intensity change of amide protons on the side arm chain and bound water intensity change on the Eu complex. Upon addition of one equivalence of calcium ions, the CEST intensity was decreased considerably from 60% to 20% for Yb complex and from 35% to 10% for the Eu complex, respectively [33].

FIGURE 21–6 Chemical structure of EuDOTA(gly)4.

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FIGURE 21–7 Zinc and calcium CEST responsive agents. CEST, Chemical exchange saturation transfer.

21.4 Conclusions and Future Perspectives The lanthanide-based responsive agent provides a new platform for molecular imaging in biological systems. To date, there are many types of responsive agents that have been designed and studied and most of these are suitable for in vitro applications. The challenge for MRI-responsive agents is to introduce them to living systems for clinical applications. This research topic is still in progress, with many opportunities to meet the ultimate potential of this frontier research field. For a T1 type agent, the enhancement of relaxivity is one of the most important factors that should be considered as MRI contrast agents are used in the millimolar range. This is an especially large challenge from the point of in vivo applications. Another important barrier that needs to be addressed is that most lanthanide-based contrast agents are unable to penetrate the cell membrane into cells. Future work on this issue should be focused on conjugating specific function groups that are capable of crossing the cell membrane. Furthermore, it would be interesting to use an MRI sensor to investigate the metabolic process inside the cell. Another issue with most responsive MRI contrast agents is that it is difficult to quantify the agent in the detecting media since the heterogeneous contrast agent would induce different contrasts in terms of the responsive mechanism. Combining MRI with another imaging modality like CT or PET may be a solution. T2ex is also a new type of imaging methodology which has been studied for some years. However, it is still limited by its intrinsic problem of negative-type contrast agents. Making ratiometric agents (T1/T2ex) for metal-responsive agents may be another method to solve this inhomogeneous problem. It can be envisioned that lanthanide-based metal-responsive agents including T1 and CEST will continuing to move toward the goal of clinical application in human disease diagnosis.

Chapter 21 • Lanthanide-Based Magnetic Resonance Imaging Metal-Responsive

479

References [1] S. Viswanathan, Z. Kovacs, K.N. Green, S.J. Ratnakar, A.D. Sherry, Alternatives to gadolinium-based metal chelates for magnetic resonance imaging, Chem. Rev. 110 (5) (2010) 29603018. [2] S. Aime, M. Botta, M. Fasano, E. Terreno, Lanthanide(III) chelates for NMR biomedical applications, Chem. Soc. Rev. 27 (1) (1998) 1929. [3] E.L. Que, C.J. Chang, Responsive magnetic resonance imaging contrast agents as chemical sensors for metals in biology and medicine, Chem. Soc. Rev. 39 (1) (2010) 5160. [4] L. Lattuada, A. Barge, G. Cravotto, G.B. Giovenzana, L. Tei, The synthesis and application of polyamino polycarboxylic bifunctional chelating agents, Chem. Soc. Rev. 40 (5) (2011) 30193049. [5] V.C. Pierre, S.M. Harris, S.L. Pailloux, Comparing strategies in the design of responsive contrast agents for magnetic resonance imaging: a case study with copper and zinc, Acc. Chem. Res. 51 (2) (2018) 342351. [6] P. Caravan, J.J. Ellison, T.J. McMurry, R.B. Lauffer, Gadolinium(III) chelates as MRI contrast agents: structure, dynamics, and applications, Chem. Rev. 99 (9) (1999) 22932352. [7] R.B. Lauffer, Paramagnetic metal complexes as water proton relaxation agents for NMR imaging: theory and design, Chem. Rev. 87 (5) (1987) 901927. [8] A.D. Sherry, Y. Wu, The importance of water exchange rates in the design of responsive agents for MRI, Curr. Opin. Chem. Biol. 17 (2) (2013) 167174. [9] S. Aime, S.G. Crich, E. Gianolio, G.B. Giovenzana, L. Tei, E. Terreno, High sensitivity lanthanide(III) based probes for MR-medical imaging, Coord. Chem. Rev. 250 (11) (2006) 15621579. [10] J.A. Peters, J. Huskens, D.J. Raber, Lanthanide induced shifts and relaxation rate enhancements, Prog. Nucl. Magn. Reson. Spectrosc. 28 (3) (1996) 283350. [11] G. Otting, Prospects for lanthanides in structural biology by NMR, J. Biomol. NMR 42 (1) (2008) 19. [12] Z. Lei, L. JingJing, L. Kai, Recent advances in gadolinium-based MRI metal responsive agent, Sci. China Technol. Sci. 61 (9) (2018) 1329. [13] M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality of calcium signalling, Nat. Rev. Mol. Cell Biol. 1 (2000) 11. [14] W.-H. Li, G. Parigi, M. Fragai, C. Luchinat, T.J. Meade, Mechanistic studies of a calcium-dependent MRI contrast agent, Inorg. Chem. 41 (15) (2002) 40184024. [15] K. Dhingra, M.E. Maier, M. Beyerlein, G. Angelovski, N.K. Logothetis, Synthesis and characterization of a smart contrast agent sensitive to calcium, Chem. Commun. 29 (2008) 34443446. [16] D.J. Waggoner, T.B. Bartnikas, J.D. Gitlin, The role of copper in neurodegenerative disease, Neurobiol. Dis. 6 (4) (1999) 221230. [17] E.L. Que, C.J. Chang, A. Smart, Magnetic resonance contrast agent for selective copper sensing, J. Am. Chem. Soc. 128 (50) (2006) 1594215943. [18] J.H. Jang, S. Bhuniya, J. Kang, A. Yeom, K.S. Hong, J.S. Kim, Cu21-responsive bimodal (optical/MRI) contrast agent for cellular imaging, Org. Lett. 15 (18) (2013) 47024705. [19] R.B. Lauffer, D.J. Parmelee, S.U. Dunham, H.S. Ouellet, R.P. Dolan, S. Witte, et al., MS-325: albumintargeted contrast agent for MR angiography, Radiology 207 (2) (1998) 529538. [20] W. Maret, Zinc biochemistry: from a single zinc enzyme to a key element of life, Adv. Nutr. 4 (1) (2013) 8291. [21] B.J. Kim, Y.H. Kim, S. Kim, J.W. Kim, J.Y. Koh, S.H. Oh, et al., Zinc as a paracrine effector in pancreatic islet cell death, Diabetes 49 (3) (2000) 367372.

480

THERANOSTIC BIONANOMATERIALS

[22] S.A. Myers, A. Nield, M. Myers, Zinc transporters, mechanisms of action and therapeutic utility: implications for type 2 diabetes mellitus, J. Nutr. Metab. 2012 (2012) 13. [23] V. Kolenko, E. Teper, A. Kutikov, R. Uzzo, Zinc and zinc transporters in prostate carcinogenesis, Nat. Rev. Urol. 10 (2013) 219. [24] A.C. Esqueda, J.A. López, G. Andreu-de-Riquer, J.C. Alvarado-Monzón, J. Ratnakar, A.J.M. Lubag, et al., J. Am. Chem. Soc. 131 (32) (2009) 1138711391. [25] J. Yu, A.F. Martins, C. Preihs, V. Clavijo Jordan, S. Chirayil, P. Zhao, et al., Amplifying the sensitivity of zinc(II) responsive MRI contrast agents by altering water exchange rates, J. Am. Chem. Soc. 137 (44) (2015) 1417314179. [26] S. Forsen, R. Hoffman, Study of moderately rapid chemical exchange reactions by means of nuclear magnetic double resonance, J. Chem. Phys. 39 (11) (1963) 28922901. [27] K.M. Ward, A.H. Aletras, R.S. Balaban, A New Class of Contrast Agents for MRI Based on Proton Chemical Exchange Dependent Saturation Transfer (CEST), J. Magn. Reson. 143 (2000) 7987. [28] S. Zhang, K. Wu, A.D. Sherry, Unusually sharp dependence of water exchange rate versus lanthanide ionic radii for a series of tetraamide complexes, J. Am. Chem. Soc. 124 (16) (2002) 42264227. [29] P.C.M. van Zijl, C.K. Jones, J. Ren, C.R. Malloy, A.D. Sherry, MRI detection of glycogen in vivo by using chemical exchange saturation transfer imaging (glycoCEST), Proc. Natl. Acad. Sci. U. S. A. 104 (11) (2007) 43594364. [30] S. Zhang, P. Winter, K. Wu, A.D. Sherry, A. Novel, Europium(III)-based MRI contrast agent, J. Am. Chem. Soc. 123 (7) (2001) 15171518. [31] S. Zhang, C.R. Malloy, A.D. Sherry, MRI thermometry based on PARACEST agents, J. Am. Chem. Soc. 127 (50) (2005) 1757217573. [32] R. Trokowski, J. Ren, F.K. Kálmán, A.D. Sherry, Selective sensing of zinc ions with a PARACEST contrast agent, Angew. Chem. Int. Ed. 44 (42) (2005) 69206923. [33] G. Angelovski, T. Chauvin, R. Pohmann, N.K. Logothetis, É. Tóth, Calcium-responsive paramagnetic CEST agents, Bioorg. Med. Chem. 19 (3) (2011) 10971105.

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively. A A10 RNA-Dox physical conjugate, 433 AAc. See Acrylic acid (AAc) Abdomen, bionanomaterials promoting healing in, 223 224 Abdominal trauma, 223 Abrasion wound, 213 Abraxane, 87 88 Acoustic contrast-enhancing bionanomaterials, 413. See also Optical contrast-enhancing bionanomaterials ACQ effects. See Aggregation-caused quenching effects (ACQ effects) Acrylic acid (AAc), 115 116 Acticoat, 219 220 Adaptive immune system, 57 59, 60f immunity modulation, 67 Adenosine triphosphate (ATP), 334 335 Adipose-derived stem cells (ADSCs), 371 Adjuvants, 241 immunostimulatory adjuvants and effect in immune response, 242t Adoptive cell transfer, 62 63 ADSCs. See Adipose-derived stem cells (ADSCs) Adsorptive transcytosis, 305 ADYNOVATE, 85 Aerosol, 15 Affinity screening strategy, 114 Age-related macular degeneration (AMD), 429 aptamers against, 429 Agglomeration effects of NPs, 9 11 Aggregation effects of NPs, 9 11 Aggregation-caused quenching effects (ACQ effects), 359 360 Aggregation-induced emission (AIE), 360 AIE-chitosan NPs, 365 AIE-SiO2 NPs, 365

AGO. See Amine-functionalized GO (AGO); Apatamer grapheme oxide (AGO) AIE. See Aggregation-induced emission (AIE) AIEgens, 360 361 AIEgen-based FL NPs, 360 fabrication of AIEgen-based nanoparticles, 361 367 with functional ligands, 364 365 functionalization of AIEgen-based NPs, 365 367, 366f enabling multifunctionality, 367 enhancing targeting efficiency, 365 366 molecular structures and optical properties, 362t into nanoparticles, 361 365, 363f Albumin-imprinting strategy, 130 132 Alendronate-functionalized CaP NPs (ALNfunctionalized CaP NPs), 158 159 Alkaline rare earth fluoride-based upconversion nanomaterials β-NaGdF4, 454 457 β-NaLuF4, 457 465 β-NaYF4, 454 calculation setup, 451 452 Alkaline-ions, 451 ALN-functionalized CaP NPs. See Alendronatefunctionalized CaP NPs (ALNfunctionalized CaP NPs) Aloe vera extract (AV extract), 219 Alzheimer’s disease, 307 AMD. See Age-related macular degeneration (AMD) Amine-functionalized GO (AGO), 176 Amino groups, 366 (3-Aminopropyl)triethoxysilane TCPSi (APTSTCPSi), 245f, 247f Anisotropic NPs, 6 7

481

482

Index

Anti-BRAF siRNA (siBraf), 433 434 Antibacterial drug, 214 215 Antibiotics, 269 Antibodies, 59 Anticancer drug-delivery system, nanoparticles as, 61 63 Antigen delivery to DCs, 61 62 Antigen-presenting cells (APCs), 57 58, 231 232 Antiinflammatory effect of bionanomaterials, 214 215 Antimicrobial agents, 96 Antioxidants, 90 91 Antithrombotic therapy, aptamers for, 429 430 Antivenom, 115 Apatamer grapheme oxide (AGO), 435 APCs. See Antigen-presenting cells (APCs) ApDCs. See Aptamer drug conjugates (ApDCs) APM. See Poly(methyl vinyl ether-alt-maleic acid)-modified APTSTCPSi (APM) Aptamer-modified graphene-FET, 439 Aptamer drug conjugates (ApDCs), 430 431 aptamer chemotherapeutic conjugates, 431 433, 432f targeted drug-delivery vehicles conjugated with aptamers, 433 434 for targeted therapies, 430 434 Aptamers, 425 applications, 428 439 aptamer micelle, 433 434 cocrystal structures of aptamers with ligands, 426 427 primary aptamer structures, 425 426 structure and complexes, 425 428, 426f structure prediction, 427 428 APTANI aptamer, 427 428 Aptasensors, 434 435, 438f APTSTCPSi. See (3-Aminopropyl)triethoxysilane TCPSi (APTSTCPSi) AR. See Aspect ratio (AR) Arc discharge, 383 Arg-Gly-Asp (RGD), 273 Artificial ECM, 221 Artificial joints, 212 213 AS1411, 428 429 AS1411-PEG-liposome (ASLP), 433 434

ASLP. See AS1411-PEG-liposome (ASLP) Aspect ratio (AR), 35, 237 Astrocytes, 306 ATP. See Adenosine triphosphate (ATP) AuNCs. See Gold nanocages (AuNCs) AuroLase, 79 81 Autophagy dysfunctions, 314 Auzel’s ET UC model, 449 AV extract. See Aloe vera extract (AV extract) B B cells, 59 B16. F10 model, 249t B16. OVA model, 249t Bacillus anthracic spores, 439 Bacillus Calmette Guerin (BCG), 63 Bacillus ribosomal protein S8, 427 Bacteria inhibition, 119 122 Barbituric acid, 475 476, 476f Bare calcium phosphate nanoparticles (Bare CaPs NP), 152f core NPs, 151 152 core shell NPs, 152 153 multilayer CaP NPs, 153 154 BBB. See Blood brain barrier (BBB) BBTB. See Blood brain tumor barrier (BBTB) BCG. See Bacillus Calmette Guerin (BCG) BCP. See Nano-biphasic calcium phosphate (BCP) BDNF. See Brain-derived neurotrophic factor (BDNF) β-lactamase, 119 120 β-NaGdF4, 454 457 β-NaLnF4, 451 453, 457 465 β-NaYF4, 451, 454 β-tricalcium phosphate (TCP), 151 Bi2S3 nanocrystals, 266 267, 353 Bioactive compounds, 297 Biocompatibility of CDs, 388 390 Biocompatible and stimuli-responsive polymer, 273 Biodegradable PLGA-loaded BP (BPQDs), 279 Biodistribution of nanoparticles, 15 16, 28 37 administration route effect, 36 37 nanoparticle effect rigidity, 36

Index

shape, 34 35 size, 32 33 surface material effect charge, 30 32 PEGylation, 28 30 Biofunctional magnetic nanomaterials functions of bionanomaterials, 342f MNPs for hyperthermia-based therapy, 349 352 for theranostic treatment, 352 354 MRI-based multimodal diagnosis, 345 349 synthesis and modification, 342 344 Biofunctionalization of QDs, 82 Biohybrid scaffolds, 173 175 Bioimaging of CDs, 388 390 Biological clearance, 38 Biological ECM, 221 Biological molecules, 324 325 Biomarkers, in vitro assays of, 326 327 Biomaterial scaffold, 310 Biomedical applications of CDs, 388 396 Biomedical cements, 222 Biomedical imaging, 122 128 MIP-NGs preparation for HAS, 131f MIPs preparation for specific membrane protein recognition, 129f MRI, 266 optical imaging, 265 PET imaging, 268 photoacoustic imaging, 268 polymeric shell, 125f procedure for preparation of SA-MIPs, 124f x-ray computed tomography imaging, 266 267 Biomedicines, synthetic receptors for bacteria inhibition, 119 122 biomedical imaging, 122 128 cancer therapy, 128 132 cell isolation, 132 134 other potentials, 135 137 toxin neutralization, 115 119 Biomolecules, 27 in vivo detection of, 327 328 Bionanomaterials, 401 acoustic contrast-enhancing, 413

483

bionanomaterial-based tissue regeneration scaffolds, 216 magnetic contrast-enhancing, 401 406 optical contrast-enhancing, 406 413 in promoting bone fracture and tendon healing, 221 223 promoting healing in abdomen, 223 224 in promoting neuron repair, 223 on wound healing, 211 213 antibacterial drug, 214 215 antiinflammatory effect, 214 215 application on skin wound healing, 218 221 due to ECM regulation, 215 216 in future, 225 modulating growth factors in wound site, 217 218 obstacles to bionanomaterial application, 224 225 supporting skin regeneration by promoting stem cell growth, 216 217 X-ray contrast-enhancing, 414 415 Biosensing, upconversion nanoprobes for, 326 328 in vitro assays of biomarkers, 326 327 in vivo detection of biomolecules, 327 328 Biosensors, 434 439 CDs as, 390 393 electrochemical detection, 435 optical detection, 435 438 other detection methods, 438 439 Biotin, 366, 425 BIS. See N,Nʹ-methylenebis(acrylamide) (BIS) 2,3-Bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl) amino)phenyl]fumaronitrile (TPETPAFN), 361, 362t, 367 4,7-Bis[4-(1,2,2-triphenylvinyl)phenyl]benzo-2,1,3thiadiazole (BTPETD), 361, 362t Black phosphorus (BP), 263 264, 279 280 singlet oxygen characterization, 280f Blood brain barrier (BBB), 31, 303 304, 307f nanobiomaterials used as imaging and diagnosing agents at BBB, 312 314 used to repair and/or regenerate BBB, 309 312

484

Index

Blood brain barrier (BBB) (Continued) role in CNS diseases, 306 308 structure and function, 305 306 Blood brain tumor barrier (BBTB), 306 307 BMCDs, 246 confocal microscopy of, 243f BMDCs. See Bone marrow-derived dendritic cells (BMDCs) BMSCs. See Bone marrow stem cells (BMSCs); Bone marrow-derived mesenchymal stem cells (BMSCs) Bone fracture, bionanomaterials in promoting, 221 223 Bone marrow stem cells (BMSCs), 199 200 Bone marrow-derived dendritic cells (BMDCs), 238 239 Bone marrow-derived mesenchymal stem cells (BMSCs), 368 Bone tissue, graphene and GO in, 168 170 Boronic acid (B(OH)2), 390 Bottom-up approaches, 381, 383 384. See also Top-down approaches microwave/ultrasonic-assisted method, 384 pyrolysis, 383 384 template-supported method, 384 Bovine serum albumin (BSA), 361 364 BP. See Black phosphorus (BP) BPEN. See N,N-bis(2-pyridyl-methyl) ethylenediamine (BPEN) BPQDs. See Biodegradable PLGA-loaded BP (BPQDs) Brain toxicity, 45 Brain tumors, 306 307 Brain-derived neurotrophic factor (BDNF), 192 193, 201 202 Brillouin zone (BZ), 451 452 Brownian relaxation mechanism, 349 351 BSA. See Bovine serum albumin (BSA) BTPETD. See 4,7-Bis[4-(1,2,2-triphenylvinyl) phenyl]benzo-2,1,3-thiadiazole (BTPETD) Bulk water proton, 477 BZ. See Brillouin zone (BZ) 5-BzdU. See 5-(N-benzyl-carboxyamide)-2'deoxyuridine (5-BzdU)

C C-dot-ZW8000 particles, 37 C-dots. See Cornell dots (C-dots) C-type lectin receptors (CLRs), 57 58, 241 C-X-C chemokine ligand 12 (CXCL12), 425, 429 C6 glioma cancer cells, 370 371 CaC. See Calcium carbonate (CaCO3) Calcium, 471 472 calcium MRI-responsive agents, 471 472, 472f calcium-responsive CEST agents, 477 CEST responsive agents, 477, 478f Calcium carbonate (CaCO3), 154 156, 268 269 Calcium chloride (CaCl2), 152 153 Calcium phosphates (CaPs), 147, 294 CaP nanoparticle-based systems bare CaPs NP, 151 154 coated CaPs NP, 154 159 therapeutics delivered using calcium phosphate NPs, 148 151 types, 151 159 CaP-based nanomaterials, 147 148 NPs, 148 149 Camptothecin (CPT), 272 273 Cancer, 83 84, 289, 342, 368 cells, 269 membranes, 248 deaths, 370 371 immunotherapy, 63 64 metastasis, 359 nanoparticles as vaccines against, 64 66 therapy, 128 132 aptamers for, 428 429 CaPs. See Calcium phosphates (CaPs) CAR-T. See Chimeric antigen receptor-expressing T cells (CAR-T) Carbon carbon-based nanoparticles, 55 57 nanomaterials, 199, 269 270 Carbon dots (CDs), 377 biomedical applications, 388 396 as biosensors, 390 393 characteristics, 379t classification and nomenclature, 378, 379f PL, 379 381, 380f spectroscopic properties, 385 387

Index

synthesis, 381 384 in vivo evaluation using rat model, 391f Carbon nanodot (CND), 378 Carbon nanotubes (CNTs), 10, 76 78, 311 312, 377 Carbon quantum dot (CQD), 378 Cardiac tissues, 173 Cartilage callus formation, 213 CAs. See Contrast agents (CAs) CASTEP code, 451 452 Cationic lipid-PLGA hybrid NPs, 242 243 CBs. See Conduction bands (CBs) CD20, 271 CD30, 271 CD52, 271 CDNs. See Cyclic dinucleotides (CDNs) CDs. See Carbon dots (CDs) CdSe-core QDs, 43 Cell behavior monitoring, 359 cell-surface receptor-mediated transcytosis, 305 imaging, 122 isolation, 132 134 membrane glycans, 122 123 migration, 359 viability tests, 156 158 Central nervous system (CNS), 187, 303 306 BBB role in, 306 308 diseases, 304 CeO2 NPs, 6 9, 17f Ceramic materials, 94 96 CEST. See Chemical exchange saturation transfer (CEST) CF composite films. See Collagen fibrin composite films (CF composite films) cGAMP. See Cyclic guanosine monophosphate (cGAMP) CH3NH3PbBr3 nanocrystals, 364 365 ChABC. See Chondroitinase ABC (ChABC) Charge of NPs, 238 239 Checkpoint inhibitors, 60 61 Chemical antibodies. See Aptamers Chemical doping, 447 Chemical exchange saturation transfer (CEST), 469

485

Chemotherapy, 269, 273, 431 Chimeric antigen receptor-expressing T cells (CAR-T), 61 Chitosan (CS), 272 273, 365 Chitosan hydrogel/nZnO bandages (CZBs), 219 Chondroitinase ABC (ChABC), 192 193 Chronic lymphocytic leukemia (CLL), 429 Ciliary-derived neurotrophic factor (CNTF), 192 193 Cisplatin, 269 Clinical translation of nanomaterials diagnostics, 76 83 CNTs, 76 78 nanoparticles, 78 82 QDs, 82 83 therapeutics, 83 93 tissue engineering, 93 101 CLL. See Chronic lymphocytic leukemia (CLL) CLRs. See C-type lectin receptors (CLRs) CN. See Coordination number (CN) CND. See Carbon nanodot (CND) CNS. See Central nervous system (CNS) CNTF. See Ciliary-derived neurotrophic factor (CNTF) CNTPEG. See Poly(ethylene glycol)-functionalized carbon nanotube (CNTPEG) CNTs. See Carbon nanotubes (CNTs) Coagulation, 213, 224 Coated CaPs NP, 154 159 LCP NPs, 154 156 polymer coating, 156 159 Collagen, 215 216 fibrils, 216 Collagen fibrin composite films (CF composite films), 171 Collective exciton effect, 379 381 Complementing nanoparticle-based therapies, 64 Computed tomography (CT), 263 264, 329, 345, 359 360, 388, 402 404, 414 Conduction bands (CBs), 449 Conductive pheyl-propanoic acid-substituted polythiophene, 396 Confocal microscopy, 90 91 of BMCDs, 243f Contrast agents (CAs), 289, 470

486

Index

Conventional downconversion luminescence nanomaterials, 321 Conventional high-efficiency lanthanide-doped upconversion nanoparticles, 322 324 Conventional organic fluorophores, 78 79 Coordination number (CN), 453 Coordination polymers. See Metal-organic frameworks (MOFs) Copper, 472 473 Copper oxide NPs, 46 Copper sulfide-loaded Fe-doped tantalum oxide (Fe-m Ta2O5@CuS), 402 404 Coprecipitation method, 343 344 Core shell engineered NPs, 448 449 interface, 450 451 quantum yields of core shell systems, 449 Cornell dots (C-dots), 79 Corrosion effects of NPs, 13 14 Covalent binding method, 365 CPT. See Camptothecin (CPT) CQD. See Carbon quantum dot (CQD) Crystallinity, 150 CS. See Chitosan (CS) CS-functionalized nanographene oxide (NGO-CS), 272 273 CT. See Computed tomography (CT) CTL. See Cytotoxic T lymphocyte (CTL) CTLA4. See Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) 64 Cu alloyed gold nanoclusters (64CuAuNCs), 33 CXCL12. See C-X-C chemokine ligand 12 (CXCL12) Cyclic diadenylate, 241 Cyclic diguanylate, 241 Cyclic dinucleotides (CDNs), 241 Cyclic guanosine monophosphate (cGAMP), 241 Cys-coated FePt NPs, 347 Cytochrome c, 265 Cytokines, 246 248, 435 Cytotoxic T cells, 59, 67 Cytotoxic T lymphocyte (CTL), 237 Cytotoxic T lymphocyte-associated antigen 4 (CTLA4), 271

Cytotoxicity, 43, 81 82 CZBs. See Chitosan hydrogel/nZnO bandages (CZBs) D Danger-associated molecular patterns (DAMPs), 231 232 DC-specific ICAM-3-grabbing nonintegrin (DCSIGN), 241 DCs. See Dendritic cells (DCs) DDSs. See Drug-delivery systems (DDSs) Deep brain stimulation, 332 334 Defective wound closure function, 214 Degradation effects of NPs, 13 14 Dendrimers, 87 Dendritic cells (DCs), 57 58, 61 enhancing immunity through delivery to, 61 Dendritic nanoparticles, 55 57 Density functional theory (DFT), 447 448 calculation method, 450 Destabilization process, 90 91 Detoxification, 119 120 DFT. See Density functional theory (DFT) Diabetes, 249 251 animal models, 250 Diabetic skin, 214 Diabetic wounds, 214 DiaCEST agents, 475 476 Diagnostic applications of aptamers, 439 1,2-Dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP), 154 156 Dioleoylphosphatidic acid (DOPA), 154 156 Discoidal polymeric nanoconstructs (DNPs), 239 Divalent zinc, 473 DNA-modified Fe3O4@Au MNPs, 343 344 DNA CND nanohybrid materials, 390 392 DNPs. See Discoidal polymeric nanoconstructs (DNPs) DO3A derivatives, 471 472 Docetaxel, 86 Donor chromophore, 406 408 DOPA. See Dioleoylphosphatidic acid (DOPA) Dorsal root ganglia (DRG), 189 191 DOTAP. See 1,2-Dioleoyl-3-trimethylammoniumpropane chloride salt (DOTAP)

Index

Double-stranded DNA (dsDNA), 272 273 Doxorubicin (DOX), 7, 61, 148, 269, 331, 392 393, 431, 433 hydrochloride, 89 90 Doxorubicin-loaded PEGylated nanographene oxide (NGO-PEG-DOX), 273 Dressings, bionanomaterials application in, 218 220 DRG. See Dorsal root ganglia (DRG) Drug-delivery systems (DDSs), 147 148, 272 273, 289, 291 292 TMDs for, 275 dsDNA. See Double-stranded DNA (dsDNA) Dual-parameter sandwich-hybridization assay, 326 327 Dye-conjugated photon upconverting nanoprobes, 321 322 Dye-labeled polymeric micelles, 292 293 Dynamic biointerface fabrication, 134 E EAE. See Experimental autoimmune encephalomyelitis (EAE) EBs. See Embryoid bodies (EBs) ECM. See Extracellular matrix (ECM) EGF. See Epidermal growth factor (EGF) EGFR. See Epidermal growth factor receptor (EGFR) EIB. See Epitope-imprinted biointerface (EIB) Electric stimulation, 211 Electrical stimulation process, 199 Electrochemical aptasensors, 435 carbonization, 383 detection, 435, 436f Electronic engineering materials, 462 Electrospinning, 188 Electrospun nanofibrous conduits, 199 Embryoid bodies (EBs), 171 172 EMU process. See Energy migration-mediated UC process (EMU process) Endocytosis, 16 Endogenous diamagnetic molecules, 475 476 Endosome, 149 Endothelial cells, 310 311

487

Energy difference, 453 454 Energy migration-mediated UC process (EMU process), 450 Energy transfer (ET), 322 324, 447 Enhanced permeability and retention (EPR), 291 292 effect, 29 30 Epidermal growth factor (EGF), 219 Epidermal growth factor receptor (EGFR), 428 429 Epitope-imprinted biointerface (EIB), 134 Epitope-imprinting epitope-imprinted synthetic receptors, 128 130 process, 133 134 EPR. See Enhanced permeability and retention (EPR) Erbium-enriched core shell nanoparticles, 322 324 Estrasorb, 86 ET. See Energy transfer (ET) Etoposide, 269 Eu/Yb-DO4A (gly)4, 477 EuDOTA(gly)4, 476 477, 477f EuIII-bound water molecule, 477 Excision wound, 213 Excretion of nanoparticles, 38 41 Exosite-II for heparin, 427 Experimental autoimmune encephalomyelitis (EAE), 249 251 Extracellular matrix (ECM), 113, 170 171, 187, 213 bionanomaterials promoting wound healing due to ECM regulation, 215 216 F 18

F-labeled molecules, 388 FA. See Folic acid (FA) Factor ELeven Inhibitory APtamer, 429 430 Factor IXa (FXIa), 429 430 FDA. See Food and Drug Administration (FDA) Fe-based magnetic bionanomaterials. See Ironbased magnetic bionanomaterials (Febased magnetic bionanomaterials) Fe-m Ta2O5@CuS. See Copper sulfide-loaded Fedoped tantalum oxide (Fe-m Ta2O5@CuS)

488

Index

Fe3O4 nanoparticles, 329 Fe3O4@Ag nanocomposites, 344 Fe3O4@MSNs-based drug-delivery system, 354 Ferrocene (Fc), 343 344 FeSe2-dispersed Bi2Se3 nanosheets, 348 349 FG sheets. See Fluorinated graphene sheets (FG sheets) Fibroblast growth factor (FGF), 217 Fibroblasts, 359 Fibronectin (FN), 132 133 Field-effect transistors, 439 FITC-doped silica NPs. See Fluorescein isothiocyanate-doped silica NPs (FITCdoped silica NPs) FL cellular tracers, 359 360 FL imaging. See Fluorescent imaging (FL imaging) Flexibility/elastic modulus of NPs, 239 Fluorescein isothiocyanate-doped silica NPs (FITC-doped silica NPs), 126 Fluorescence method, 78 Fluorescence resonance energy transfer (FRET), 392 393, 406 408 FRET-based ratiometric fluorescent nanoprobe, 406 408 Fluorescence-labeled silica nanoparticles, 265 Fluorescent imaging (FL imaging), 263 264, 359 360 Fluorescent proteins (FPs), 359 360 Fluoride-based lanthanide nanocrystals, 324 Fluorinated graphene sheets (FG sheets), 176 Fluorophores, 78 79, 386 5-Fluorouracil (5-FU), 269 FN. See Fibronectin (FN) Folate-decorated matrix materials, 365 366 Folic acid (FA), 265 Food and Drug Administration (FDA), 79 FPs. See Fluorescent proteins (FPs) FRET. See Fluorescence resonance energy transfer (FRET) 5-FU. See 5-Fluorouracil (5-FU) Fucose (Fuc), 122 123 Functional CNS, 306 FXIa. See Factor IXa (FXIa)

G g-C3N4 QDs. See Single-layered graphitic C3N4 QDs (g-C3N4 QDs) G-quadruplex structures, 425 G-rich quadruplex. See Guanine-rich quadruplex (G-rich quadruplex) Gadolinium (Gd), 297 chelates, 266 ions, 455, 470 Gadolinium MRI-responsive agents, 470 474 relaxivity change mechanism, 470 471 survey of Gd-based MRI sensors, 471 474 inner sphere number modulation of water molecules, 471 473 rotational tumbling time modulation, 473 474 Gadolinium neutron capture therapy (GdNCT), 297, 298f Gadolinium-based MRI contrast agent, 469 Gastrointestinal tract (GI tract), 15 GBM. See Glioblastoma (GBM) GCGR. See Glucagon receptor (GCGR) Gd-diethylenetriaminepentaacetic acid (GdDTPA), 297 Gd-DO3A derivative Ca sensor, 472 Gd-DO3A derivative copper sensors, 472 473 Gd-DTPA. See Gd-diethylenetriaminepentaacetic acid (Gd-DTPA) Gd-DTPA-loaded polymeric micelles, 293 294 Gd31-doped WS2 nanosheet (Gd-WS2), 274 GdNCT. See Gadolinium neutron capture therapy (GdNCT) Gel fillers, 201 Gelatin, 87 88 Gelatin methacryloyl (GelMA), 171 172 Gelators, 97 98 GelMA. See Gelatin methacryloyl (GelMA) Gemcitabine, 148 GEMs. See Germ-line transgenic, conditional transgenic models (GEMs) Gene delivery, 272 273 TMDs for, 275 Gene therapy, 271 Germ-line transgenic, conditional transgenic models (GEMs), 248

Index

GFP. See Green fluorescent protein (GFP) GI tract. See Gastrointestinal tract (GI tract) Gibbs Thomson effect, 9 Glass transition temperature (Tg), 36 GlcA. See Glucuronic acid (GlcA) Glioblastoma (GBM), 439 Glucagon receptor (GCGR), 430 Glucose transporter-1 (GLUT-1), 304 Glucuronic acid (GlcA), 122 125 Glycan-imprinted gold nanorod, 130 GM-CSF. See Granulocyte macrophage colony stimulating factor (GM-CSF) GNSs. See Gold nanostars (GNSs) GO-PEI. See Polyethylenimine branch modified grapheme oxide (GO-PEI) GO-PNIPAM complex. See PNIPAM functionalized GO complex (GO-PNIPAM complex) Gold (Au) clusters, 348 glyconanoparticles, 313 Gold nanocages (AuNCs), 269 270 Gold nanoparticles (AuNPs), 30, 42, 64, 79, 82, 240f, 312 313, 392 393 AuNPs @ PDA, 347 plasmonic, 269 270 Gold nanorod (AuNR), 79 81, 130, 269 270 Gold nanostars (GNSs), 437 438 GQD. See Graphene quantum dot (GQD) Granulation tissue, 213 Granulocyte macrophage colony stimulating factor (GM-CSF), 246 248 Granulomatosis, 17 Graphene, 165 166, 179f, 263 264, 272 273 gene and drug delivery, 272 273 graphene-based materials, 165 166, 171 as phototherapeutic agent, 273 in tissue engineering, 166 applications of graphene and GO based on properties, 167f properties and applications in, 166 178 Graphene oxide (GO), 165 166, 263 264, 272 sheets, 381 in tissue engineering, 166

489

applications of graphene and GO based on properties, 167f properties and applications in, 166 178 Graphene quantum dot (GQD), 378 surface/edge state, 381 in vitro evaluation, 392f Graphite flakes, 382 Graphitic carbon nitride (g-C3N4), 263 264, 278 Graphitic nanomaterials, 377 Green fluorescent protein (GFP), 359 360, 388 390 Ground-state formation enthalpy calculation, 453 wavefunction, 463 464 Guanine-rich quadruplex (G-rich quadruplex), 428 429 H H-bond. See Hydrogen bond (H-bond) HA. See Hydroxyapatite (HA) HA/SF composites. See HA/silk fibroin composites (HA/SF composites) HA/silk fibroin composites (HA/SF composites), 168 170 Hair follicle stem cells, 216 217 Hairpin loops, 425 HAuNSs. See Hollow gold nanospheres (HAuNSs) HD1 aptamer, 427 HD22 aptamer, 427 Heart toxicity, 45 Heavily doped upconversion nanoparticles, 322 324 HEMA. See 2-Hydroxyethyl methacrylate (HEMA) Hemostasis. See Coagulation Heparin, 429 430 HepG2-specific aptamer, 433 Hermatite (α-Fe2O3), 313 314 Heterogeneity, 344 Heterogeneous assay, 326 327 Heterojunction induced band offsets, 449 “Hidden” epitope-imprinting method, 128 130 HIF-1ɑ. See Hypoxia inducible factor-1ɑ (HIF-1ɑ) HIV-1 Tat, 366 367 hNSCs. See Human neural stem cells (hNSCs) Hollow gold nanospheres (HAuNSs), 269 270

490

Index

Homogeneous assay, 326 327 HPV. See Human papilloma virus (HPV) HPMA. See Poly[N-(2-hydroxypropyl) methacrylamide] (HPMA) HSA. See Human serum albumin (HSA) HTC. See Hydrothermal carbonization (HTC) Hubbard-U, 448, 451 Human cells, 246 248 Human neural stem cells (hNSCs), 173 Human papilloma virus (HPV), 232 Human serum albumin (HSA), 470 471 Human VEGF (hVEGF), 126 128 Hyaluronic acid (HA), 151 scaffolds, 310 311 Hydroactive dressing, 220 Hydrogels, 97 101, 99f, 100f, 189 191, 310 Hydrogen bond (H-bond), 425 426 Hydrogen peroxide (H2O2), 344 Hydrophobic interaction, 331 Hydrothermal carbonization (HTC), 383 384 Hydrothermal method, 324, 343 344 Hydroxyapatite (HA), 94 96, 168 170 Hydroxycarbamide, 269 2-Hydroxyethyl methacrylate (HEMA), 120 122 Hydroxyurea, 269 Hyperbaric oxygen therapy, 211 Hyperthermia-based therapy, MNPs for, 349 352 combined therapy, 352 MHT, 349 351 PTT, 351 352 Hypoxia inducible factor-1ɑ (HIF-1ɑ), 216 217 I i.v. injection. See Intravenous injection (i.v. injection) IA. See Itaconic acid (IA) Ibuprofen, 192 193 ICAM-1. See Intercellular adhesion molecule I (ICAM-1) ICG. See Indocyanine green (ICG) ICP-MS. See Inductively coupled plasma mass spectrometry (ICP-MS) IFN-γ. See Interferon-γ (IFN-γ) Ig. See Immunoglobulins (Ig) Imaging agents, TMDs as, 274

Imaging contrast agents, 401 Iminodiacetate, 472 473 Immunofluorescence staining, 189 191 Immunoglobulins (Ig), 59 Immunostimulation, 234f, 249 251 for cancer, 248 249 Immunotherapy, 60 61, 231 233, 233t nanoparticles, 63 64 as anticancer drug-delivery system, 61 63 enhancing immunity through delivery to DCs, 61 system design, 235 243 nanotechnology for, 234 235 in vitro assessment, 244 248 human cells, 246 248 murine cells, 244 246 in vivo models and efficacy, 248 251 immunomodulation, 249 251 immunostimulation for cancer, 248 249 In vitro assays of biomarkers, 326 327 In vitro assessment, 244 248 human cells, 246 248 murine cells, 244 246 In vitro cell cultures, 390 In vitro cell tracing, 368 369 In vitro selection or evolution, 423 In vivo bioimaging using upconversion nanoparticles, 328 330 multimodel bioimaging, 329 330 near-infrared light-based optical imaging, 328 329 In vivo detection of biomolecules, 327 328 In vivo long-term cell tracing, 370 371 In vivo models and efficacy, 248 251 immunomodulation, 249 251 immunostimulation for cancer, 248 249 In vivo multicolor imaging, 329 Indocyanine green (ICG), 347 Inductively coupled plasma mass spectrometry (ICP-MS), 28, 40 Inflammation, 213 Inflammatory response, 192 193 Inhalation, 45 Innate immune system, 57 58 Innate immunity modulation, 67

Index

Inner sphere number modulation of water molecules, 471 473 Inorganic materials, 147 148 Inorganic nanoparticles (INPs), 4 7, 78 79 and interest in medicine, 3 8 physicochemical modifications determining biodistribution and fate, 15 18 physicochemical modifications in physiological environments, 8 14, 10f effects of adsorption of (macro)molecules, 11 13 effects of agglomeration and aggregation, 9 11 effects of corrosion and degradation, 13 14 Inorganic upconversion nanocrystals, 322 324 INPs. See Inorganic nanoparticles (INPs) Intercellular adhesion molecule I (ICAM-1), 29 30 Interferon-γ (IFN-γ), 435 Interleukins (ILs) IL-1α, 213 IL-1β, 213 IL-4, 59, 246 248 IL-8, 216 217 Intracellular uptake and release mechanism, 149 Intravenous injection (i.v. injection), 12, 15, 32 Intrinsic nanomaterial characteristics, 239 240 IONC@Au-PEG nanoplatform, 351 352 Ionic interaction, 425 426 IONPs. See Iron oxide nanoparticles (IONPs) Ipilimumab, 60 61 Iron (Fe), 344 Iron oxide (Fe3O4), 402 404 nanoclusters with enhanced magnetization, 405 406 nanomaterials, 402 404 Iron oxide nanoparticles (IONPs), 66, 79 82, 266 Iron-based magnetic bionanomaterials (Fe-based magnetic bionanomaterials), 401 Isothermal titration calorimetry, 158 159 Isothiocyanate (NCS), 365 Itaconic acid (IA), 120 122 K Keloid, 214, 220 221 Kidney toxicity, 43 44

491

L Laceration wound, 213 Laminin, 196 197 Laminin-attached graphene, 176 Lanthanide (Ln) lanthanide-based MRI contrast agents, 469 lanthanide-doped nanocrystals, 328 329 nanomaterials, 334 335 nanoparticles, 326 327 upconversion nanomaterials, 321 322 upconversion nanoparticles, 329 330 materials, 447 spectroscopy, 448 Laser ablation, 381 382 Latent or superimposed microbial infections, 214 Layered double hydroxide (LDH), 65 66, 263 264, 280 281 LCP NPs. See Lipid-coated CaP NPs (LCP NPs) LDH. See Layered double hydroxide (LDH) Lewis lung cancer cells (LLC cells), 249t Ligand cocrystal structures of aptamers with, 426 427 engineering, 324 325 ligand-free lanthanide-doped nanoparticles, 334 335 ligand-mediated liposomal targeting systems, 92 93 Linear MIP (LMIP), 120 Linear polymers (LPs), 117 Lipid-coated CaP NPs (LCP NPs), 154 156, 155f Lipidocolloid technology (TLC), 220 TLC NOSF, 220 Lipopolysaccharide (LPS), 62, 231 232 Liposomal nanoparticles, 55 57 Liposome with hydrogel core of silk fibroin (SF-LIP), 217 Liposomes, 88 93, 433 434 Liver, 15 toxicity, 42 43 LLC cells. See Lewis lung cancer cells (LLC cells) LMIP. See Linear MIP (LMIP) Localized surface plasmon resonance (LSPR), 266 267, 351

492

Index

Long-term cell tracing with AIEgen-based fluorescent nanoparticles, 367 371. See also AIEgens in vitro cell tracing, 368 369 in vivo long-term cell tracing, 370 371 Longitudinal relaxation time (T1), 402 Longitudinal relaxivity, 470 471, 471f, 473 LPs. See Linear polymers (LPs) LPS. See Lipopolysaccharide (LPS) LRET. See Luminescence resonance energy transfer (LRET) LSPR. See Localized surface plasmon resonance (LSPR) Luminescence, 406. See also Photoluminescence (PL) mechanism of upconversion nanoparticles, 322 324 nanoparticles, 321 Luminescence resonance energy transfer (LRET), 326 327 Lung toxicity, 45 46 Lymphocytes, 239 240 Lyophilized siRNAs, 151 152 Lysosomes, 17 M Macrophages, 306 Macugen, 429 Maghemite (γ-Fe2O3), 313 314 Magnetic contrast-enhancing bionanomaterials, 401 406, 402f magnetomotive imaging, 405 406 MPI, 404 MRI, 401 404 Magnetic hybrid nanomaterials (MHNs), 341 343 Magnetic hyperthermia therapy (MHT), 349 351 Magnetic nanomaterials, 402 Magnetic nanoparticles (MNPs), 341 hybridization, 343 344 for hyperthermia-based therapy, 349 352 for theranostic treatment, 352 354 Magnetic particle imaging (MPI), 401, 404 Magnetic resonance (MR), 263 264

Magnetic resonance imaging (MRI), 77, 263 264, 266, 289, 312, 329, 342, 359 360, 388, 401 404, 469 MRI-based multimodal diagnosis, 345 349 MRI CT bimodal diagnosis, 345 347 MRI ECT bimodal diagnosis, 347 polymeric micelles for tumor, 293 294, 295f Magnetite (Fe3O4), 313 314 Magnetization transfer (MT), 474 475 Magnetomotive imaging, 405 406 Magnetomotive photoacoustic imaging (mmPA imaging), 405 406 Major histocompatibility complex (MHC), 57 58, 231 232 Manganese dioxide (MnO2), 266 nanosheets, 263 264, 280 281 Manganese oxide (MnO), 402 Manganese-based magnetic bionanomaterials, 401 Mannose (Man), 122 123 Matrix metalloproteases (MMPs), 402 404 Matrix metalloproteinases, 307 308 MC38 model, 249t MDDCs. See Monocyte-derived dendritic cells (MDDCs) Medical imaging, 281 Melanoma tumor model, 65 6-Mercaptopurine, 269 Mesenchymal stem cells (MSCs), 168 170, 193 194, 216 217 Mesoporous nanostructures, 263 Mesoporous silica nanoparticles (MSNs), 35, 354 Mesoporous-silica-coated upconversion nanoparticles, 321 322 Metal halides, 77 metal-based nanoparticles, 55 57 metal-mediated base pairs, 343 344 NPs, 12 Metal oxide NPs, 12 Metal-organic frameworks (MOFs), 263 264, 276 278, 384 Metalloproteinase-9 (MMP-9), 313 Methacrylic groups, 97 98

Index

Methacryloyl substituted recombinant human tropoelastin (MeTro), 171 172 Methicillin-resistant Staphylococcus aureus (MRSA), 119 120 Methotrexate (MTX), 158 159, 269 Methylene blue, 343 344 MeTro. See Methacryloyl substituted recombinant human tropoelastin (MeTro) MHC. See Major histocompatibility complex (MHC) MHNs. See Magnetic hybrid nanomaterials (MHNs) MHT. See Magnetic hyperthermia therapy (MHT) MI. See Myocardial infarction (MI) Micro-OCT imaging (μOCT imaging), 409 Microbubble, 268 269 Microemulsion method, 343 344 Microglia, 306 Microneedles (MNs), 409 Microwave/ultrasonic-assisted method, 384 Mineral bone deposition, 213 MIP-FITC-NPs. See Monosaccharide-imprinted FITC-modified NPs (MIP-FITC-NPs) MIP-QDs. See Molecularly imprinted polymercoated QDs (MIP-QDs) MIPs. See Molecularly imprinted polymers (MIPs) MMP-9. See Metalloproteinase-9 (MMP-9) mmPA imaging. See Magnetomotive photoacoustic imaging (mmPA imaging) MMPs. See Matrix metalloproteases (MMPs) MNPs. See Magnetic nanoparticles (MNPs) MNs. See Microneedles (MNs) MODCs. See Monocyte-derived DCs (MODCs) MOFs. See Metal-organic frameworks (MOFs) Molecular imaging, 289 Molecular imprinting process, 114 Molecularly imprinted polymer-coated QDs (MIPQDs), 125 126 Molecularly imprinted polymers (MIPs), 114, 118 MIP-based synthetic receptors, 128, 136 137 Molybdenum disulfide (MoS2), 274 Monoclonal antibodies, 63, 232 Monocyte-derived DCs (MODCs), 246 248 surface-dependent immunogenicity of PSi particles, 247f

493

Monocyte-derived dendritic cells (MDDCs), 239 240 Monocytes, 213 Mononuclear phagocytic system (MPS), 27 28 clearance, 39 40 Monosaccharide-imprinted FITC-modified NPs (MIP-FITC-NPs), 126 MoS2. See Molybdenum disulfide (MoS2) MP-SERS. See Myoglobin-and polydopamineengineered SERS (MP-SERS) MPI. See Magnetic particle imaging (MPI) MPS. See Mononuclear phagocytic system (MPS) MR. See Magnetic resonance (MR) MRI. See Magnetic resonance imaging (MRI) mRNAs, 64 65 MRSA. See Methicillin-resistant Staphylococcus aureus (MRSA) MS. See Multiple sclerosis (MS) MS-325, 473 MSCs. See Mesenchymal stem cells (MSCs) MSNs. See Mesoporous silica nanoparticles (MSNs) MT. See Magnetization transfer (MT) MTX. See Methotrexate (MTX) MUC1. See Mucin 1 protein (MUC1) Mucin 1 protein (MUC1), 390 Multi-walled CNTs (MWCNTs), 76 Multicolor-emitting upconversion nanoparticles, 332 Multifunctional MHNs, 351 353 Multilayer CaP NPs, 153 154 Multimodality imaging, polymeric micelles for tumor, 294 297, 296f Multimodel bioimaging, upconversion nanoparticles for, 329 330 Multiple sclerosis (MS), 307, 430 Murine cancer model, 249t cells, 244 246 EMT6 breast cancer model, 34 35 MWCNTs. See Multi-walled CNTs (MWCNTs) Myocardial infarction (MI), 171 172 Myoglobin-and polydopamine-engineered SERS (MP-SERS), 408 409

494

Index

N N,N-bis(2-pyridyl-methyl)ethylenediamine (BPEN), 473 474 N,Nʹ-methylenebis(acrylamide) (BIS), 115 116 N-3-aminopropyl methacrylamide, 115 116 N-acetylneuraminic acid (NANA), 125 126 5-(N-benzyl-carboxyamide)-2'-deoxyuridine (5BzdU), 428 429 N-doped CDs, 384, 386 387 N-isopropylacrylamide (NIPAm), 115 116 N-tert-butylacrylamide (TBAm), 115 116, 135 136 NaGdF4, 452 NANA. See N-acetylneuraminic acid (NANA) Nano-based scaffolds, 222 Nano-biphasic calcium phosphate (BCP), 222 Nano-oligosaccharide factor (NOSF), 220 Nano-scaled theranostic agent, 309 Nanobiomaterials, 304 305 used as imaging and diagnosing agents at BBB, 312 314 gold nanoparticles, 312 313 quantum dots, 313 SPIONs, 313 314 used to repair and/or regenerate BBB, 309 312 carbon nanotubes, 311 312 scaffolds, 310 311 Nanobubbles, 413 Nanofibers, 189, 195 196 hydrogel, 189 191, 201 Nanofibrous scaffolds, 188 Nanomaterials, 8, 27, 281 282, 308 309 clinical translation diagnostics, 76 83 therapeutics, 83 93 tissue engineering, 93 101 design for neural tissue engineering, 188 nanomaterial-based diagnostics, 83 for peripheral nerve injury repair, 194 204 for SCI repair, 188 194 Nanomedicine, 8 9, 15, 18 19, 27 28, 211, 212f, 388 Nanoparticles (NPs), 8, 27, 55 57, 77 82, 114, 147, 202, 212 213, 234 235, 388 390, 448 adjuvants, 241

immunostimulatory adjuvants and effect in immune response, 242t AIEgens into, 361 365 antigen position, 242 243 biodistribution, 28 37 challenges, 66 68 modulating innate and adaptive immunity, 67 challenges and perspectives, 46 47 characteristics, 67 68 charge, 238 239 classes, 56f complementing nanoparticle-based therapies, 64 as diagnostics, 66 effects of agglomeration and aggregation of, 9 11 entering body, 15 16 entering cells, 16 17 excretion, 38 41 mononuclear phagocytic system clearance, 39 40 renal clearance, 41, 44f flexibility/elastic modulus, 239 for immunotherapy, 60 61 as immunotherapy, 63 64 nanoparticle-based drugs, 47 parameters for rational design of nanosystems, 236f properties and impact on immune system, 56f shape, 237, 238f size, 235 237 surface chemistry and roughness, 239 241 therapy, 57 58 toxicity, 28, 42 46 brain toxicity, 45 heart toxicity, 45 kidney toxicity, 43 44 liver toxicity, 42 43 lung toxicity, 45 46 as vaccines against cancer, 64 66 Nanopowders, 11 Nanosafety, 15, 19 Nanoscale metal-organic frameworks (NMOFs), 266

Index

Nanosized PEGylated GO (NGO-PEG), 273 Nanosystems, 234 Nanotechnology, 47, 75, 212 213, 250, 352 353, 377 for immunotherapy, 234 235 nanotechnology-based theranostics, 321 Nanovaccines, 234, 248, 250 murine cancer model in efficacy assessment of, 249t Nanowires (NWs), 435 Natural extracellular matrix components, 310 Natural materials, 196 197 Natural polymers, 94 96 NBD. See Nitrobenzoxadiazole (NBD) NCS. See Isothiocyanate (NCS) Near-infrared (NIR), 6 7, 78, 178, 294 297, 351 biological nanoprobes, 328 329 fluorescence, 377 light, 269 270 light-activated photodynamic therapy, 331 332 light-based optical imaging, 328 329 light-mediated luminescence nanoparticles, 327 328 light-mediated optogenetic therapy, 332 334 light-triggered drug delivery, 330 331 molecules, 265 NIR-excitable upconversion nanoparticles, 321 322, 326 327, 332 334 photons, 321 322 Néel relaxation mechanism, 349 351 Negative-pressure wound therapy, 211 Neomycin, 426 427 Nephrotoxicity, 43 Nerve conduits design for repairing PNI, 194 201 Nerve growth factor (NGF), 192 193 Nerve guidance conduits (NGCs), 194 Nervous system, 187 Neural stem cells (NSCs), 189, 311 Neural tissue engineering, 172, 187 design of nanomaterials, 188 nanomaterials for peripheral nerve injury repair, 194 204 for SCI repair, 188 194 Neuro-Spinal Scaffold, 96

495

Neurodegenerative diseases, 306 307 Neurological diseases, 307 308 Neurons, 187 bionanomaterials in promoting neuron repair, 223 Neurotrophic factors, 192 193 Neurotrophin-3 (NT-3), 192 193 Neurovascular unit, 303 304 Neutrophils, 213 NF-κB. See Nuclear factor-κB (NF-κB) NGCs. See Nerve guidance conduits (NGCs) NGF. See Nerve growth factor (NGF) NGO-CS. See CS-functionalized nanographene oxide (NGO-CS) NGO-PEG. See Nanosized PEGylated GO (NGOPEG) NGO-PEG-DOX. See Doxorubicin-loaded PEGylated nanographene oxide (NGOPEG-DOX) NIP-QDs. See Nonimprinted controls QDs (NIPQDs) NIPAm. See N-isopropylacrylamide (NIPAm) NIR. See Near-infrared (NIR) Nitrobenzoxadiazole (NBD), 123 124 NLRs. See NOD-like receptors (NLRs) NMOFs. See Nanoscale metal-organic frameworks (NMOFs) NOD-like receptors (NLRs), 57 58 Noncovalent ApDCs, 433 Noncovalent binding method, 361 365 Nondrug-formulated nanoparticles, 63 64 Nonimprinted controls QDs (NIP-QDs), 125 126 Noninvasive cell tracking, 359 Noninvasive in vivo cell tracing, 370 Nonionizing laser pulses, 410 412 Normal wound repair, 213 NOSF. See Nano-oligosaccharide factor (NOSF) NOX-A12, 429 NPs. See Nanoparticles (NPs) NrGO. See Single-layered nano-rGO (NrGO) NSCs. See Neural stem cells (NSCs) NT-3. See Neurotrophin-3 (NT-3) Nuclear factor-κB (NF-κB), 241 Nucleic acids, 149 synthesis, 269

496

Index

Nucleolin, 425, 428 429 Nucleotide aptamers, 423 applications, 428 439 structure and complexes, 425 428 NWs. See Nanowires (NWs) O OAm. See Oleylamine (OAm) Obesity, 430 Ochratoxin A (OTA), 435 OCT. See Optical coherence tomography (OCT) 1-Octadecene (ODE), 345 346 Oleylamine (OAm), 345 346 Oligo(poly(ethylene glycol) fumarate) (OPF), 171 172 OligoCalc, 427 428 Oligofectamine, 154 156 OPF. See Oligo(poly(ethylene glycol) fumarate) (OPF) Opsonin, 28 29 Opsonosis, 28 29 Optical coherence tomography (OCT), 406, 409 Optical contrast-enhancing bionanomaterials, 406 413, 406f FRET, 406 408 luminescence, 406 OCT, 409 PAI, 410 413 Raman imaging, 408 409 Optical detection, 435 438 Optical imaging, 328 329 polymeric micelles for, 292 293 Optogenetics, 332 334 Organic AIEgen-based NPs, 360 fluorogens, 360 molecules, 359 360 OTA. See Ochratoxin A (OTA) Oxaliplatin-linked CQDs, 396 P PAA. See Polyacrylic acid (PAA) Paclitaxel (PTX), 297 PAI. See Photoacoustic imaging (PAI)

PAMAM. See Poly(amidoamine) dendrimers (PAMAM) PAMP. See Pathogen associated molecular pattern (PAMP) PANi. See Polyaniline (PANi) Paramagnetic chemical exchange saturation transfer (paraCEST), 469, 474 477 zinc and calcium CEST responsive agents, 477, 478f Partial density of states (PDOSs), 457 Particle size, 151 Particle-induced cell death, 334 335 PAT. See Photoacoustic tomography (PAT) Pathogen associated molecular pattern (PAMP), 57 58 Pattern recognition receptors (PRRs), 57 58 PBMCs. See Peripheral blood monocytes (PBMCs) PC. See Protein corona (PC) PCE. See Poly(citric acidoctanediol-polyethylene glycol) (PCE) PCL. See Poly(ε-caprolactone) (PCL) PCR. See Polymerase chain reaction (PCR) PD-L1 antibody, 60 61 PDA@Fe3O4, 343 344 PDOSs. See Partial density of states (PDOSs) PDs. See Polymer dots (PDs) PDT. See Photodynamic therapy (PDT) PEDOT. See Poly(3,4-ethylenedioxythiophene) (PEDOT) PEG. See Polyethylene glycol (PEG) PEG-coated spinel-phase Fe3O4 nanoparticles, 404 Pegaptanib. See Macugen pEGFR-targeted Ba2GdF7 NPs, 347 PEGylation, 28 30, 39 40, 91 PEGylated Fe@Fe3O4 MHNs, 351 352 PEGylated SPIONs, 314 of proteins, 87 88 PEI. See Polyethylenimine (PEI) Pericytes, 306 Peripheral blood monocytes (PBMCs), 246 248 Peripheral nerve injury (PNI), 187, 194 nanomaterials for repair, 194 204 nanomaterials combined with cell therapy, 202 204

Index

nanomaterials combined with growth factors, 201 202 nerve conduits design, 194 201 Peripheral nervous system (PNS), 187 PET. See Positron emission tomography (PET) PFODBT. See Poly(2,7-(9,9-dioctylfluorene)-alt4,7-bis(thiophen-2-yl)benzo-2,1,3thiadiazole) (PFODBT) Phenol-soluble modulin α3 (PSMα3), 115 116 Phosphate ions, 384 Phosphatidylcholine/cholesterol liposomes, 30 Phospholipase A2 (PLA2), 116 117 Phosphor luminescence technique, 447 Phosphorescence, 386 387, 389f Photoacoustic imaging (PAI), 78, 263 264, 268, 359 360, 406, 410 413 Photoacoustic signal, 410 412 Photoacoustic tomography (PAT), 294 297 Photodynamic therapy (PDT), 64, 82, 128 130, 263 264, 270 271, 321 322, 396 Photoluminescence (PL), 377, 385 386. See also Luminescence of CDs, 379 381, 380f quenching, 381 Photon upconversion, 322 324 photon upconversion-mediated medical therapy, 330 334 NIR light-activated photodynamic therapy, 331 332 NIR light-mediated optogenetic therapy, 332 334 NIR light-triggered drug delivery, 330 331 Photosensitizers (PS), 321 322 Phototherapeutic agent, graphene as, 273 Photothermal agents, TMDs as, 275 276 Photothermal OCT (PTOCT), 409 Photothermal therapy (PTT), 64, 263 264, 269 270, 349, 351 352 Physicochemical modifications of inorganic NPs determining biodistribution and fate, 15 18 biodistribution, 15 16 long-term effects, 17 18 subcellular localization, 16 17 in physiological environments, 8 14, 10f π π stacking, 425 426

497

PL. See Photoluminescence (PL) PLA. See Polylactic acid (PLA) PLA2. See Phospholipase A2 (PLA2) Plasma cells, 59 Platelet-rich fibrin (PRF), 222 Platinum (Pt), 297 PLCP. See Polycation liposome-encapsulated CaP NPs (PLCP) PLEGRIDY, 85 PLGA. See Poly(lactic-co-glycolic acid) (PLGA) PLGA PEG folate. See Poly[lactide-coglycolide] PEG folate (PLGA PEG folate) PLLA. See Poly(l-lactide) (PLLA) PMA. See Poly(methyl acrylate) (PMA) PMMA. See Poly-methylmetacrylate (PMMA) pMMUS. See Pulsed magnetomotive ultrasound imaging (pMMUS) PNI. See Peripheral nerve injury (PNI) pNIPAAm. See Poly(N-isopropylacrylamide) (pNIPAAm) PNIPAM. See Polyi(N-isopropylacrylamide) (PNIPAM) PNIPAM functionalized GO complex (GOPNIPAM complex), 273 PNS. See Peripheral nervous system (PNS) Point-of-care biosensors, 76 Poly-methylmetacrylate (PMMA), 222 Poly(2,7-(9,9-dioctylfluorene)-alt-4,7-bis (thiophen-2-yl)benzo-2,1,3-thiadiazole) (PFODBT), 412 413 Poly(3,4-ethylenedioxythiophene) (PEDOT), 173, 197 199 Poly(amidoamine) dendrimers (PAMAM), 87, 272 273 Poly(citric acidoctanediol-polyethylene glycol) (PCE), 170 171 Poly(ethylene glycol)-functionalized carbon nanotube (CNTPEG), 173 Poly(l-lactide-co-ε-caprolactone) [P(LLA-CL)], 189 Poly(l-lactide) (PLLA), 192 193 Poly(lactic-co-glycolic acid) (PLGA), 168 170, 191, 242 243, 271 Poly(methyl acrylate) (PMA), 36

498

Index

Poly(methyl vinyl ether-alt-maleic acid)-modified APTSTCPSi (APM), 247f Poly(N-isopropylacrylamide) (pNIPAAm), 178 Poly(styrene) (PS), 36 Poly(ε-caprolactone) (PCL), 170 171 Poly[lactide-co-glycolide] PEG folate (PLGA PEG folate), 365 366 Poly[N-(2-hydroxypropyl)methacrylamide] (HPMA), 85 86 Polyacrylic acid (PAA), 31 Polyamine-functionalized CDs, 390 Polyaniline (PANi), 197 199 Polycaprolactone-based scaffolds, 310 311 Polycation liposome-encapsulated CaP NPs (PLCP), 154 156 Polyclonal antibodies, 115 Polyethylene glycol (PEG), 16, 27 28, 79 81, 85, 156, 266 267, 291 292, 310, 324 325, 348 349, 402, 429 Polyethylene oxide, 85 Polyethylenimine (PEI), 31, 156 Polyethylenimine branch modified grapheme oxide (GO-PEI), 272 273 Polyethylenimine-modified UnTHCPSi (UnP), 247f Polyi(N-isopropylacrylamide) (PNIPAM), 273 Polylactic acid (PLA), 94 96 Polymer coating, 156 159, 157f nanodrugs, 84 85 polymer-based bio-nanotherapeutics, 84 85 Polymer dots (PDs), 378 Polymerase chain reaction (PCR), 153 154 Polymeric micelles, 86, 291 292, 294 for tumor theranostics, 291f, 297 imaging modalities for cancer imaging, 290t polymeric micelles for tumor imaging, 292 297 Polymerics nanomaterials, 84 88 Polypyrrole (PPy), 197 199 Polysorbate 20, 42 Polyvinyl alcohol (PVA), 170, 222 Polyvinylpyrrolidone (PVP), 31, 266 267 Porous silicon NPs (PSi NPs), 239 240 interaction, 245f

Porphyrinic bacteriochlorophyll lipid microbubble, 268 269 Positron emission tomography (PET), 28, 263 264, 268, 289, 312, 329 PPy. See Polypyrrole (PPy) PRF. See Platelet-rich fibrin (PRF) Primary aptamer structures, 425 426 Pristine Fe3O4 NPs, 343 344 Proinflammatory cytokines, 213, 307 Proliferation, 213 Prostheses, 212 213 Protein corona (PC), 9 Protein tyrosine kinase 7, 431 Proteins, 11, 87 88, 148 149 PrPSc, 435 PRRs. See Pattern recognition receptors (PRRs) PS. See Photosensitizers (PS); Poly(styrene) (PS) PS-loaded NaYF4Yb/Er@SiO2 nanoparticles, 331 332 Pseudoknot, 425 PSi NPs. See Porous silicon NPs (PSi NPs) PSMα3. See Phenol-soluble modulin α3 (PSMα3) PTOCT. See Photothermal OCT (PTOCT) PTT. See Photothermal therapy (PTT) PTX. See Paclitaxel (PTX) Pulsed magnetomotive ultrasound imaging (pMMUS), 405 406 Purmorphamine, 192 193 PVA. Poly(vinyl alcohol) (PVA);. See Polyvinyl alcohol (PVA) PVP. See Polyvinylpyrrolidone (PVP) Pyrolysis, 383 384 Q QCM. See Quartz crystal microbalance (QCM) QDs. See Quantum dots (QDs) QS. See Quorum sensing (QS) Qtracker655, 367 368, 370 371 Quantum confinement effect, 379 381 Quantum dots (QDs), 31, 76, 82 83, 122, 125 126, 265, 313, 321, 359 360 Quantum electrodynamical calculations, 406 408 Quantum yield (QY), 361 of core shell systems, 449 Quartz crystal microbalance (QCM), 115 116

Index

Quercetin, 90 91 Quorum sensing (QS), 120 QY. See Quantum yield (QY) R RA. See Rheumatoid arthritis (RA) Rac1, 428 429 Radiation therapy (RT), 263 264, 349 Radiationless mechanism, 406 408 Radical oxygen species (ROS), 42 43 Radical polymerization, 97 98 Radio-nuclide imaging, 263 264 Radioactive Au nanostructures, 34 35 Radiolabeling techniques, 28 Radionuclide rhenium-188 (188Re), 294 297 Radiotherapy, 273 Raman imaging, 406, 408 409 Raman scattered light, 408 409 Rapamycin, 62 63 Rare earth (RE), 447 RE-based fluoride system, 448 Rational design of nanosystems, 235, 236f RE. See Rare earth (RE) Reactive oxygen species (ROS), 270 271, 307, 408 409 Reactive phase, 213 Real-time noninvasive imaging methodology, 409 Recombinant human epidermal growth factor (rhEGF), 219 Reduced GO (rGO), 170 171, 263 264, 272 Reepithelialization. See Proliferation Regulatory T cells (Treg), 59 Relaxation effect, 470 Relaxivity change mechanism, 470 471 Remodeling phase, 213 Renal clearance, 41 Reparative phase, 213 Responsive probes, 469 Retinoic acid, 192 193 Reverse thermal gelation, 97 98 RGD. See Arg-Gly-Asp (RGD) rGO. See Reduced GO (rGO) rGO into polyacrylamide (rGO/PAAm), 170 171 rhBMPs, 94 96

499

rhEGF. See Recombinant human epidermal growth factor (rhEGF) Rheumatoid arthritis (RA), 249 251 drug-loaded mineralized pegylated particles, 251f Rhodamine-B-labeled chitosan nanoparticles, 31 RIG-I-like receptors, 57 58 Rituximab, 61 RNA aptamers, 426 427 “Roll & seal” method, 199 200 ROS. See Radical oxygen species (ROS); Reactive oxygen species (ROS) Rotational tumbling time modulation, 473 474 Roughness of NPs, 239 241 RRKJ method, 451 452 RT. See Radiation therapy (RT) S SA. See Sialic acid (SA) Scaffolds, 94 96, 310 311 Scanning electron microscopy (SEM), 189 191 Scar formation, 220 221 Schwann cells, 194, 201, 204 SCI. See Spinal cord injury (SCI) “Screened pseudo-charge potential” model, 462 Second diffusion-controlled method, 98 SELEX. See Systematic evolution of ligands by exponential enrichment (SELEX) Self-absorption, 361 Self-assembling peptides, 191, 223 Self-supporting graphene hydrogel film, 178 SEM. See Scanning electron microscopy (SEM) Semiconductor polymer nanomaterials (SPN), 412 413 Semiconductor quantum dots (SQDs), 377 Semiconductor-based nanocrystals, 377 Sensitizer, 322 324 SERS. See Surface-enhanced Raman scattering (SERS) SF-bFGF-LIP, 217 SF-LIP. See Liposome with hydrogel core of silk fibroin (SF-LIP) sgc8 (DNA aptamer), 431 Shape of NPs, 237, 238f Shell core type, 344

500

Index

Sialic acid (SA), 122 123 siBraf. See Anti-BRAF siRNA (siBraf) Silica, 79 nanoparticles, 82, 263 silica-based nanoparticles, 55 57 silica-coated upconversion nanoparticles, 324 325 Silicon dioxide (SiO2), 79 Silver, 218 sulfadiazine, 214 Silver nanoparticles (AgNPs), 45, 214 217 Single-layered graphitic C3N4 QDs (g-C3N4 QDs), 278 Single-layered nano-rGO (NrGO), 273 Single-photon emission computed tomography (SPECT), 28, 289, 345 Single-stranded DNA (ssDNA), 272 273 Single-stranded oligonucleotides (ss oligonucleotides), 423 Single-walled CNTs (SWCNTs), 76 SWCNT-FET, 439 Size of NPs, 235 237 Skin regeneration by promoting stem cell growth, 216 217 Skin wound healing application, 218 221 bionanomaterials application in dressings, 218 220 in suture fabrication, 220 use of bionanomaterials and keloid, 220 221 SMAD3-antisense oligonucleotides, 221 Small-molecule drugs, 148 Soft graphene nanofibers, 191 Sol gel technique, 97 98, 151 152 Solomon's Bergen Morgan equation, 470 471 Solvothermal carbonization (STC), 383 384 Solvothermal methods of coprecipitation and decomposition, 324 method, 343 344 sp2-hybridization, 166 167 SPECT. See Single-photon emission computed tomography (SPECT) Spinal cord injury (SCI), 187 nanomaterials for repair, 188 194 design, 189 191

nanomaterials combined with cell therapy, 193 194 nanomaterials combined with growth factors, 192 193 SPIO. See Superparamagnetic iron-oxide (SPIO) SPIONs. See Superparamagnetic iron-oxide nanoparticles (SPIONs) SPN. See Semiconductor polymer nanomaterials (SPN) SPR. See Surface plasmon resonance (SPR) SQDs. See Semiconductor quantum dots (SQDs) ss oligonucleotides. See Single-stranded oligonucleotides (ss oligonucleotides) ssDNA. See Single-stranded DNA (ssDNA) STC. See Solvothermal carbonization (STC) Stem cells, 166, 204, 359 stem cell-based therapy, 368 Steric stabilization, 67 68 Stimulator of interferon genes (STING), 241 243 STING. See Stimulator of interferon genes (STING) Stober method, 79 Strawberry-like Fe3O4 Au NPs, 344 Streptomycin, 426 427 Subcellular localization, 16 17 Super resolution microscopy, 7 Superparamagnetic iron-oxide (SPIO), 77 78, 404 NPs, 79 81 Superparamagnetic iron-oxide nanoparticles (SPIONs), 293 294, 308 309, 313 314, 388 Superparamagnetic materials, 266 Surface area-to-volume ratio, 211 212 chemistry of NPs, 239 241 modifications, 16 passivation, 390 392 quenching effect, 448 surface/edge state of GQD, 381 Surface plasmon resonance (SPR), 6 7, 79, 120, 435 biosensors, 437 438 SPR-based aptasensors, 437 438, 437f Surface-enhanced Raman scattering (SERS), 122 123, 377, 408 409

Index

Surface-to-volume ratio, 448 Surgical operations, 223 Suture fabrication, bionanomaterials application in, 220 SWCNTs. See Single-walled CNTs (SWCNTs) Synthetic polymers, 94 97 materials, 222 Synthetic receptors, 115 for biomedicines, 115 137 preparation, 114f Systematic evolution of ligands by exponential enrichment (SELEX), 423, 424f T T cell receptor (TCR), 59 T cells, 59, 62 T helper 1 cell (Th1 cell), 59 T helper 2 cell (Th2 cell), 59 Targeted drug-delivery vehicles conjugated with aptamers, 433 434 Tat. See Transactivator of transcription (Tat) TBAm. See N-tert-butylacrylamide (TBAm) TC. See Tetracycline (TC) TCP. See β-tricalcium phosphate (TCP) TCPP. See Tetrakis(4-carboxyphenyl)porphyrin (TCPP) TCPSi. See Thermally carbonized PSi (TCPSi) TCR. See T cell receptor (TCR) TD. See 2,1,3-Thiadiazole (TD) Telomerase, 269 TEM. See Transmission electron microscopy (TEM) Template-supported method, 384 Tendon healing, bionanomaterials in promoting, 221 223 TEOS. See Tetraethyl orthosilicate (TEOS) Tetra-modal imaging agent, 348 349 Tetracycline (TC), 435 Tetraethyl orthosilicate (TEOS), 361 364 Tetrahydrofuran (THF), 361 Tetrakis(4-carboxyphenyl)porphyrin (TCPP), 276 Tetramethylammonium hydroxide (TMAOH), 345 346 Tetraphenylethene (TPE), 361, 362t TPE-CS NPs, 368 TPE-FN nanocrystals, 364

501

TGF-β1/Smad signal transduction pathway, 217 Th1 cell. See T helper 1 cell (Th1 cell) THCPSi. See Thermally hydrocarbonized PSi (THCPSi) Theranostic 2D nanomaterials, 271 281 black phosphorus, 279 280 graphene and derivatives, 272 273 metal-organic frameworks, 276 278 transition metal dichalcogenides, 274 276 Theranostic agents, 308 309 Theranostic biomaterials for BBB, 305 308 explaining theranostic agents, 308 309 nanobiomaterials using to repair and/or regenerate BBB, 309 312 used as imaging and diagnosing agents at BBB, 312 314 Theranostic carbon dots, 393 396. See also Carbon dots (CDs) Theranostic nanomedicines, 3 4 Theranostic NPs, 93 Theranostic payloads biomedical imaging, 265 269 therapeutics, 269 271 Theranostic treatment, MNPs for, 352 354 Therapeutic nanomaterials, 83 93 liposomes, 88 93 polymerics, 84 88 Therapeutic nanoparticles, 55 57 Therapeutical nucleotide aptamers, 428 430 against age-related macular degeneration, 429 for antithrombotic therapy, 429 430 for cancer therapies, 428 429 against other diseases, 430 Therapeutics chemotherapy, 269 delivery using CaP NPs factors affecting drug release rates, 149 151 intracellular uptake and release mechanism, 149 therapeutics type, 148 149 gene therapy, 271 PDT, 270 271 PIT, 269 270

502

Index

Thermally carbonized PSi (TCPSi), 245f, 246 248, 247f Thermally hydrocarbonized PSi (THCPSi), 245f NPs, 239 240 Thermally oxidized PSi (TOPSi), 245f, 246 248, 247f NPs, 239 240 Thermoacoustic imaging, 410 412 ThermoDox, 91 92 THF. See Tetrahydrofuran (THF) 2,1,3-Thiadiazole (TD), 361 Thioflavin-S (ThS), 273 Three-dimension (3D) bioprinting, 99 100 graphene films, 173 localization and concentration of nanomaterials, 404 printing, 96 3D-DART webserver, 427 428 3D-printed BCP/PVA/PRF scaffolds, 222 Three-dimensional graphene foams (3D-GF), 171 Thromboses, 429 430 ThS. See Thioflavin-S (ThS) Tight junctions (TJs), 303 304 Time-dependent Schrödinger equation, 463 464 Tissue engineering, 93 101, 166. See also Neural tissue engineering applications of graphene and GO based on properties, 167f hydrogels, 97 101 properties and applications in, 166 178 chemical properties and applications, 175 177 electrical properties and applications, 172 175 mechanical properties and applications, 168 172 other properties and applications, 178 scaffolds, 94 96 skeletal tissue regeneration, 95f Titania NPs, 82 TJs. See Tight junctions (TJs) TLC. See Lipidocolloid technology (TLC) TLRs. See Toll-like receptors (TLRs) TLS11a-GC, 433

tLyp1 peptide hybrid nanoparticle, 64 TMAOH. See Tetramethylammonium hydroxide (TMAOH) TMDs. See Transition metal dichalcogenides (TMDs) TNF-α. See Tumor necrosis factor-α (TNF-α) Toll-like receptors (TLRs), 57 58, 62, 231 232 ligands, 241 TLR3-agonist polyinosinic polycytidylic acid, 62 Top-down approaches, 381 383. See also Bottom-up approaches arc discharge, 383 electrochemical carbonization, 383 laser ablation, 381 382 TOPSi. See Thermally oxidized PSi (TOPSi) Toxicity, 55 57 of nanoparticles, 42 46 studies of upconversion nanoparticles, 334 335 Toxin neutralization, 115 119 TPE. See Tetraphenylethene (TPE) TPE-FN. See 2-(2-(4-(1,2,2-Triphenylvinyl) phenyl)-4H-chromen-4-ylidene] malononitrile (TPE-FN) TPETPAFN. See 2,3-Bis(4-(phenyl(4-(1,2,2triphenylvinyl)phenyl)amino)phenyl] fumaronitrile (TPETPAFN) TPP. See Tripolyphosphate (TPP) TprA receptor, 120 Traditional chemotherapeutic drugs, 29 30 Transactivator of transcription (Tat), 366 Tat-TPETPAFN NP-labeled ADSCs, 371 Tat-TPETPAFN NPs, 367, 370 371 Transition metal dichalcogenides (TMDs), 263 264, 274 276 for gene and drug delivery, 275 as imaging agents, 274 as photothermal agents, 275 276 Transmission electron microscopy (TEM), 40, 402 404 Transverse relaxation time (T2), 402 Trap states, 380, 386 Trastuzumab, 61 Traumatic brain injury, 307 308 Treg. See Regulatory T cells (Treg)

Index

3-(4-(1,2,2-Triphenylvinyl)phenoxy)propan-1amine, 364 365 2-(2-(4-(1,2,2-Triphenylvinyl)phenyl)-4Hchromen-4-ylidene]malononitrile (TPEFN), 361 364 Tripolyphosphate (TPP), 365 Tully’s surface hopping method, 463 464 Tumor cells, 60 61, 351 352 Tumor imaging, polymeric micelles for, 292 297 magnetic resonance imaging, 293 294, 295f multimodality imaging, 294 297, 296f optical imaging, 292 293 Tumor microenvironment-responsive polymeric micelles, 292 293 Tumor necrosis factor-α (TNF-α), 213, 435 Tumor therapy, 263 264, 273 274, 279 Tunable PL, 386 Two-dimensional nanomaterials (2D nanomaterials), 263 264, 264f, 271 281 future perspectives, 281 282 theranostic, 271 281 theranostic payloads, 265 271 U UC luminescence. See Upconversion luminescence (UC luminescence) UCNPs. See Upconversion nanoparticles (UCNPs) UCPL. See Upconversion photoluminescence (UCPL) Ultrasmall superparamagnetic iron oxide (USPIO), 79 81 Ultrasonic imaging, 413 Ultrasound imaging, 268 269 Ultrathin 2D zirconium MOF (PCN-222) nanosheet, 276 278 Ultraviolet (UV), 265 absorption, 385 light irradiation, 361 Undecylenic acid-modified THCPSi (UnTHCPSi), 245f, 247f UnP. See Polyethylenimine-modified UnTHCPSi (UnP) UnTHCPSi. See Undecylenic acid-modified THCPSi (UnTHCPSi)

503

Upconversion luminescence (UC luminescence), 327 328, 447 for biosensing, 326 328 intensity and efficiency, 447 448 tuning, 325 Upconversion nanocrystals, 322 324 Upconversion nanoparticles (UCNPs), 6 7, 265, 321 322, 447 biocompatible, 325f design considerations, 322 325 electronic structural engineering, 448 lanthanid doped, 31 luminescence mechanism, 322 324 photon upconversion-mediated medical therapy, 330 334 synthesis and surface engineering, 324 325 toxicity studies, 334 335 in vivo bioimaging using, 328 330 Upconversion photoluminescence (UCPL), 386 Upconversion-based nanocarriers, 330 331 Upconversion-mediated optogenetics, 332 334 Usnic acid (Fe3O4@UA), 216 217 USPIO. See Ultrasmall superparamagnetic iron oxide (USPIO) UV. See Ultraviolet (UV) V Vaccines against cancer, nanoparticles as, 64 66 4f Vacuum referred binding energy (4f-VRBE), 463 Valence band (VB), 451 Valence band maximum (VBM), 451 Van der Waals force, 425 426 Vascular endothelial growth factor (VEGF), 126 128, 135 136, 152 153, 201 202, 306 307 Vascular endothelial growth factor receptor 2 (VEGFR2), 65 66 Vascular endothelial growth factor-165 (VEGF165), 429 VB. See Valence band (VB) VBM. See Valence band maximum (VBM) VEGF. See Vascular endothelial growth factor (VEGF) Visible light, 265 Vroman effect, 11 12

504

Index

W Water residence lifetime, 470 471 Waveband, 351 WO3 x nanodots, 266 267 Wound healing, 220 221 bionanomaterials on, 211 213 antibacterial drug, 214 215 antiinflammatory effect, 214 215 due to ECM regulation, 215 216 in future, 225 modulating growth factors in wound site, 217 218 scope in clinical practice, 218 224 supporting skin regeneration by promoting stem cell growth, 216 217 characteristics, 213 214 Wounds, 211, 213 WS2, 274, 276

X X-ray computed tomography imaging, 266 267 X-ray contrast-enhancing bionanomaterials, 414 415. See also Optical contrastenhancing bionanomaterials X-ray CT, 289 Z Zinc CEST responsive agents, 477, 478f zinc-activated MRI contrast agents, 473 474, 474f zinc-responsive agents, 473 474 Zinc phthalocyanine (ZnPc), 402 404 ZnGa2O4Cr0.004 (ZGC), 406, 407f Zwitterionic QDs, 31